WO2023074911A1 - 新規べと病抵抗性遺伝子を有するホウレンソウ植物 - Google Patents
新規べと病抵抗性遺伝子を有するホウレンソウ植物 Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/12—Leaves
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/02—Amaranthaceae or Chenopodiaceae, e.g. beet or spinach
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Definitions
- the present invention relates to spinach plants having genes that provide resistance to broad-spectrum downy mildew, and methods for producing the same.
- This application claims priority based on Japanese Patent Application No. 2021-178897 filed in Japan on November 1, 2021, the content of which is incorporated herein.
- Plant varieties generally include fixed species and hybrid first-generation (hereinafter referred to as "F1") varieties, and F1 varieties are widespread in major crops.
- the F1 variety grows vigorously due to hybrid vigor (heterosis).
- hybrid vigor heterosis
- the F1 cultivar has significant advantages such as fast growth and high yield, and is expected to have improved environmental adaptability such as resistance to pests and cold and heat resistance.
- the F1 cultivar exhibits extremely high phenotype uniformity because it has the same genotype even though it is heterozygous. This increases the marketability of the product.
- useful traits controlled by dominant genes in the parents of the F1 variety can be accumulated, rapid breeding becomes possible.
- Non-Patent Document 2 The search for new resistant materials is very important in spinach breeding in order to respond to the continuous emergence of new races.
- a search for downy mildew-resistant genetic material is being conducted not only for cultivated species but also for wild species. For example, by the Center for Genetic Resources, the Netherlands (CGN), in 2008, Spinacia turkestanica, a wild spinach, and in 2011, Spinacia tetrandra, a wild spinach. A search for disease-resistant genetic material was carried out. Also, in 2011, Correll et al. announced that six types of genes called RPF control known downy mildew resistance (Non-Patent Document 2).
- RPF1 to RPF10 RPF11 (Patent Document 1), RPF12 (Patent Document 2), RPF13 (Patent Document 3), RPF14 (Patent Document 4), RPF15 (Patent Document 5), R6 (Patent Document 6), R15 (Patent Document 7), etc.
- Spinach with high downy mildew resistance can be produced by introducing a downy mildew-resistant gene derived from wild spinach into a cultivated spinach, Spinacia oleracea L. (Patent Document 8). .
- a single resistance gene that confers resistance to all named Pfs races (Pfs1 to Pfs19), UA1014 type, Be2105B type, and PV2144 type is currently unknown. Therefore, in order to obtain resistance to all known Pfs races using known resistance genes, it is necessary to combine multiple resistance genes.
- the object of the present invention is to provide a single dominant downy mildew resistance gene that confers resistance to at least all of Pfs1 to Pfs19, UA1014 type, Be2105B type, and PV2144 type.
- the present inventors have found that on the third chromosome of Spinacia tetrandra strain RNR140003, resistance to at least all of Pfs1 to Pfs19, UA1014 type, Be2105B type, and PV2144 type.
- a new dominant downy mildew-resistant gene was found to exist, and the gene was named RPF-SK1 gene.
- interspecific crossing between the wild spinach having the downy mildew resistance gene and the cultivated spinach is performed to obtain an F1 individual having downy mildew resistance, and the F1 individual is further crossed with the cultivated spinach.
- the present inventors have found that spinach plants having downy mildew resistance attributed to the downy mildew-resistant gene and traits close to those of cultivated species can be grown, and have completed the present invention.
- the present invention is as follows. [1] having a downy mildew-resistant RPF-SK1 gene in at least one allele;
- the RPF-SK1 gene is (a) the SNP identified by chr3_1215815 is cytosine, (b) the SNP identified by chr3_1215855 is thymine; (c) the SNP identified by chr3_1216014 is cytosine; (d) the SNP identified by chr3_1216093 is guanine, the SNP identified by chr3_1216094 is guanine, and the SNP identified by chr3_1216095 is adenine; (e) the SNP identified by chr3_1216288 is adenine, or (f) the SNP identified by chr3_1216291 is guanine, Downy mildew resistant spinach plant.
- X locus is "the site occupied by the X gene in the chromosome”. Therefore, in the present invention and the specification of the present application, "a spinach plant having an X locus” is a spinach plant having a site occupied by the X gene in the chromosome, and is synonymous with "a spinach plant having an X gene”. be.
- the downy mildew-resistant spinach plant according to the present invention includes a spinach plant in which a chromosome fragment containing the RPF-SK1 locus has replaced the region corresponding to the fragment in chromosome 3, a translocation, etc. spinach plants into which a chromosomal fragment containing the RPF-SK1 locus has been introduced outside the region corresponding to the RPF-SK1 locus in chromosome 3 of Spinasia tetrandra.
- Chromosomal fragments containing the RPF-SK1 locus include, for example, nucleic acids represented by SEQ ID NOs: 3, 4, 5, and Table 1.
- the introduction of the RPF-SK1 gene into the genomic DNA can be carried out by cross-breeding using Spinacia tetrandra as the parent strain, or by genetic modification methods such as genome editing.
- the downy mildew-resistant spinach plant according to the present invention is an interspecific hybrid plant between Spinacia tetrandra and a spinach plant other than Spinacia tetrandra, and has downy mildew resistance derived from the RPF-SK1 gene.
- derived from plants that have "Interspecific hybrid plants” include, in addition to plants produced by interspecies crossing of species belonging to the genus Spinasia, somatic cell hybrid plants resulting from cell fusion between different species of plants of the genus Spinasia, and plants of the genus Spinasia. Also included are grafted hybrid plants obtained by grafting between different species.
- an interspecific hybrid plant between Spinasia tetrandra and a spinach plant other than Spinasia tetrandra includes not only the interspecific hybrid plant but also progeny of the interspecific hybrid plant.
- progeny of a spinach plant means, in addition to progeny obtained by intraspecific crossing of the spinach plant, an individual obtained as a parent line of the spinach plant and its progeny, and the spinach plant. It includes somatic hybrid plants and their progeny obtained by cell fusion between cells and plant cells of other cultivars, and individuals obtained by grafting the spinach plant as a rootstock or scion and their progeny. "Progeny” includes both individuals resulting from intraspecific and interspecific hybrids.
- individual obtained from a (plant) parental line means an individual obtained by intraspecific crossing, interspecific crossing, cell fusion, or grafting using the plant as a parental line.
- the spinach plant to be used as a material for producing the downy mildew-resistant spinach plant according to the present invention is not particularly limited as long as it has the RPF-SK1 gene possessed by Spinacia tetrandra line RNR140003.
- Spinacia tetrandra strains include progeny strains inheriting the RPF-SK1 gene from RNR140003.
- the downy mildew-resistant spinach plant according to the present invention may be the progeny of an F1 individual obtained by interspecific crossing between Spinasia tetrandra and a spinach plant other than Spinasia tetrandra.
- a spinach plant other than Spinasia tetrandra For example, spinachia tetrandra and cultivar spinach plants are crossed to obtain F1 seeds, and plant individuals grown from the obtained F1 seeds are backcrossed or selfed with cultivar spinach plant individuals to obtain seeds. Then, individuals having the RPF-SK1 gene are selected from plant individuals grown from the obtained seeds.
- Downy mildew-resistant spinach is obtained by repeatedly crossing the selected plant individuals with self-fertilized or cultivated spinach plants and selecting individuals having the RPF-SK1 gene from progeny obtained by the crossing. A fixed seed of the plant can be obtained. Selfing, crossing with cultivar spinach plants, cultivation, and collection of seeds can be carried out according to conventional spinach cultivation methods.
- Individuals having the RPF-SK1 gene can be selected from progeny obtained by crossing, for example, by examining downy mildew resistance. Specifically, Pfs of each race is inoculated to the leaves of test plant individuals to be examined for downy mildew resistance, and the presence or absence of downy mildew disease is examined. If the test plant does not develop downy mildew, it is evaluated as being resistant to the inoculated race. From progeny obtained by crossing, plant individuals having resistance to at least all races represented by downy mildew races Pfs1 to Pfs19, UA1014 type, Be2105B type, and PV2144 type are defined as individuals having the RPF-SK1 gene. be selected as
- Individuals having the RPF-SK1 gene can be selected from progeny obtained by crossing, for example, by examining whether the genomic DNA contains a chromosomal fragment containing the RPF-SK1 gene. Specifically, DNA markers in the RPF-SK1 locus of spinach plants are used for identification.
- a DNA marker is one that can detect differences in DNA sequences on chromosomes that can distinguish between chromosome fragments derived from different breeds.
- DNA markers include SNP (Single Nucleotide Polymorphism) markers, SSR (Simple Sequence Repeats) markers, and RFLP (Restriction Fragment Length Polymorphism) markers. be done.
- DNA markers can be detected by a conventional method. For example, using DNA extracted from each plant as a template, a nucleic acid amplification reaction is performed using primers and a polymerase that can specifically hybridize with a specific SNP or SSR, and the presence or absence of an amplified product is determined using electrophoresis or the like. Each polymorphism can be detected and identified. In addition, each polymorphism can be identified by detecting the pattern of DNA fragments by electrophoresis or the like after treating the DNA extracted from each individual plant with a restriction enzyme.
- Primers that can specifically hybridize with specific SNPs and SSRs are selected according to the nucleotide sequence of the genomic DNA of spinach and the nucleotide sequences of the SNPs and SSRs to be detected using commonly used primer design tools and the like. can be designed by conventional methods and synthesized by methods well known in the art.
- chr3_1216093, chr3_1216094, and chr3_1216095 have a sequence consisting of three bases that is a base sequence specific to the RPF-SK1 gene, and the combination of these three SNPs is a DNA used for selecting individuals having the RPF-SK1 gene. become a marker.
- SNP bases can be identified using, for example, KASP (Kompetitive Allele Specific PCR) genotyping assay (manufactured by LGC Genomics).
- KASP Kompetitive Allele Specific PCR genotyping assay
- the bases of these SNPs can also be identified by PCR-amplifying the region containing chr3_1215815 to chr3_1216291 using the genomic DNA extracted from the test spinach plant as a template, and identifying the base sequence of the resulting amplification product. can be done.
- spinach plants having a fragment containing the RPF-SK1 locus of Spinasia tetrandra line RNR140003 homozygously or heterozygously have chr3 — 1215815 in at least one allele.
- the SNP identified is cytosine
- the SNP identified by chr3_1215855 is thymine
- the SNP identified by chr3_1216014 is cytosine
- the three consecutive SNPs identified by chr3_1216093 to chr3_1216095 are guanine-guanine-adenine
- the SNP identified by chr3 — 1216288 is adenine
- the SNP identified by chr3 — 1216291 is guanine.
- the downy mildew-resistant spinach plant according to the present invention preferably has traits other than downy mildew resistance, particularly traits as an agricultural product, that are the same as or similar to cultivated spinach.
- the traits of agricultural crops include taste, leaf shape, yield, ease of cultivation, and the like.
- the "cultivated traits” means traits suitable for agricultural crops.
- the downy mildew-resistant spinach plant according to the present invention preferably has at least one downy mildew-resistant gene other than the RPF-SK1 gene.
- the RPF-SK1 gene alone can confer resistance to a wider range of races than known resistance genes, but it also has other downy mildew resistance genes to break through resistance. It can also be expected to contribute to suppressing the appearance of
- the downy mildew-resistant gene other than the RPF-SK1 gene is not particularly limited, and examples thereof include the RPF1 gene to RPF15 gene, R6 gene, R15 gene and the like in the case of heterozygotes.
- the downy mildew-resistant spinach plant according to the present invention may be a whole plant or a part of the plant. That is, the downy mildew-resistant spinach plant according to the present invention includes all of the spinach plant having the RPF-SK1 gene, the above-ground part of the spinach plant, and the tissue of the spinach plant.
- a downy mildew-resistant spinach plant according to the present invention also includes cells obtained from a tissue of a spinach plant having the RPF-SK1 gene. Tissues can include embryos, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons, and hypocotyls.
- the cells are plant cells that have spinach plant genomic DNA and that have the RPF-SK1 gene.
- a spinach plant cell having the RPF-SK1 gene may be a single cell or a cell mass composed of a plurality of cells.
- the spinach plant cells having the RPF-SK1 gene may be collected from any tissue of the spinach plant, or may be a cell mixture in which cells collected from different tissues are mixed. Such tissues include those listed above. Among them, single cells derived from leaf blade or petiole tissue are preferred.
- the downy mildew resistance of the downy mildew-resistant spinach plants according to the present invention exhibits dominant expression. Therefore, by crossing interspecifically or intraspecifically with the downy mildew-resistant spinach plant according to the present invention as a parent line, a new spinach plant strain resistant to downy mildew derived from the RPF-SK1 gene is cultivated. becomes possible.
- the method for screening a downy mildew-resistant spinach plant according to the present invention examines whether a test spinach plant has the RPF-SK1 gene, and if it is determined to have the RPF-SK1 gene, the test spinach plant. Select as downy mildew resistant spinach plants.
- whether or not a test spinach plant has the RPF-SK1 gene is determined by DNA markers at or near the RPF-SK1 locus, or by strongly combining these DNA markers. Judgment is based on the genotype of the linked DNA markers.
- the genotype of the DNA marker at or near the RPF-SK1 locus or the DNA marker strongly linked to these DNA markers has the RPF-SK1 gene, such as the RNR140003 strain, and is susceptible to downy mildew If it is of the same genotype as the resistant Spinacia tetrandra, the test spinach plant is selected as a downy mildew-resistant spinach plant.
- DNA markers for determining the presence or absence of the RPF-SK1 gene include the SNPs listed in Table 1. Among them, the SNP identified by chr3_1215815, the SNP identified by chr3_1215855, the SNP identified by chr3_1216014, the SNP identified by chr3_1216093, the SNP identified by chr3_1216094, the SNP identified by chr3_1216095, and chr3_121628 of the spinach plant at 8
- One or more selected from the group consisting of the identified SNP, the SNP identified by chr3_1216291, and SNPs that are strongly genetically linked to these SNPs are DNAs for determining the presence or absence of the RPF-SK1 gene Suitable as a marker.
- the SNP identified by chr3_1215815 is cytosine
- the SNP identified by chr3_1215855 is thymine
- the SNP identified by chr3_1216014 is cytosine.
- the SNP identified by chr3_1216093 is guanine
- the SNP identified by chr3_1216094 is guanine
- the SNP identified by chr3_1216095 is adenine
- the SNP identified by chr3_1216288 is adenine
- the presence or absence of the RPF-SK1 gene can be more easily determined using the kit.
- Selection for downy mildew resistant spinach plants can be done at discretion.
- the forward primer and reverse primer for PCR-amplifying the region containing chr3_1215815 to chr3_1216291 in the genomic DNA of the spinach plant can be included in the kit used to select the spinach plant having the RPF-SK1 gene. .
- a primer set for identifying the genotype of the SNP identified by chr3_1215815 of spinach plants a primer set for identifying the genotype of the SNP identified by chr3_1215855, and identifying the genotype of the SNP identified by chr3_1216014 a primer set for identifying the genotypes of three consecutive SNPs identified by chr3_1216093, chr3_1216094, and chr3_1216095; a primer set for identifying the genotype of the SNP identified by chr3_1216288;
- a kit containing a set of primers for identifying the genotype of the SNP identified by chr3_1216291 is useful for identifying the genotype of these SNPs for determining the presence or absence of the RPF-SK1 gene in test spinach plants.
- the method for predicting downy mildew resistance of a spinach plant according to the present invention examines whether the test spinach plant has the RPF-SK1 gene, and if it is determined to have the RPF-SK1 gene, the test spinach plant are likely to be downy mildew resistant.
- whether or not a test spinach plant has the RPF-SK1 gene is determined by DNA markers at or near the RPF-SK1 locus, or with these DNA markers. Judgment is based on the genotype of strongly linked DNA markers.
- the genotype of the DNA marker at or near the RPF-SK1 locus or the DNA marker strongly linked to these DNA markers has the RPF-SK1 gene, such as the RNR140003 strain, and is susceptible to downy mildew
- the test spinach plant has the RPF-SK1 gene, and has the RPF-SK1 gene such as the RNR140003 strain, and is resistant to downy mildew
- the genotype is different from that of Spinacia tetrandra, it is determined that the test spinach plant does not have the RPF-SK1 gene.
- DNA markers for determining the presence or absence of the RPF-SK1 gene include the SNPs listed in Table 1. For example, in at least one allele, (a) the SNP identified by chr3_1215815 is cytosine, (b) the SNP identified by chr3_1215855 is thymine, (c) the SNP identified by chr3_1216014 is cytosine.
- the SNP identified by chr3_1216093 is guanine
- the SNP identified by chr3_1216094 is guanine
- the SNP identified by chr3_1216095 is adenine
- the SNP identified by chr3_1216288 is adenine
- the test spinach plant carries the RPF-SK1 gene and is likely to be downy mildew resistant.
- the primer set for identifying the genotype of the DNA marker for judging the presence or absence of the RPF-SK1 gene and the kit thereof may be the same as those described above.
- ⁇ Method for producing downy mildew-resistant spinach plant By crossing spinach plants having the RPF-SK1 gene as materials, downy mildew resistance derived from the RPF-SK1 gene is introduced into spinach plants that do not have the RPF-SK1 gene to develop a novel downy mildew. Resistant spinach plants can be produced.
- the RPF-SK1 gene which is a single dominant resistance gene, can be easily introduced into any line and can accelerate the development of spinach varieties.
- the use of a spinach plant homozygous for the RPF-SK1 gene as a parent line facilitates the breeding of cultivars having resistance to a wider range of races.
- the RPF-SK1 gene can be combined in the same haplotype with other single dominant resistance alleles at different loci than the RPF-SK1 locus.
- the method for producing downy mildew-resistant spinach plants according to the present invention is a method that utilizes crossbreeding using a spinach plant having the RPF-SK1 gene as a parent line. Specifically, a first crossing step of crossing a spinach plant having the RPF-SK1 gene with any spinach plant, and the F1 individuals obtained by the first crossing step are selfed, backcrossed, or A second crossing step of obtaining a segregating population by performing interspecific crossing or intraspecific crossing with a spinach plant different from the parent line used in the first crossing step, and spinach plants having the RPF-SK1 gene from the segregating population. and a selection step of selecting. Selfing, backcrossing, interspecific crossing, intraspecific crossing, cultivation of spinach plants, and harvesting of seeds can be carried out by conventional spinach cultivation methods.
- the spinach plant having the RPF-SK1 gene used as the parent line in the first crossing step Spinasia tetrandra can be used, and the downy mildew-resistant spinach plant according to the present invention can also be used.
- the spinach plant having the RPF-SK1 gene to be used as a parent line should be a downy mildew-resistant spinach plant that has both the RPF-SK1 gene and traits as a cultivated species. preferable.
- Any spinach plant used as a parent line in the first crossing step and any spinach plant used as a parent line for interspecific or intraspecific cross in the second crossing step are not particularly limited, and RPF-SK1 Various spinach plants that do not have the gene can be used as appropriate.
- a downy mildew-resistant spinach plant that has the RPF-SK1 gene and also has traits as a cultivated species and a cultivated spinach plant that does not have the RPF-SK1 gene are used as parent lines.
- Any spinach plant used as a parent line in the first crossing step, or any spinach plant used as a parent line for interspecific or intraspecific cross in the second crossing step, downy mildew resistance other than the RPF-SK1 gene It is preferably a spinach plant having at least one gene, and more preferably a cultivar spinach plant having at least one downy mildew-resistant gene other than the RPF-SK1 gene.
- a novel spinach line containing both the RPF-SK1 gene and other downy mildew-resistant genes is created by using a spinach plant having at least one or more downy mildew-resistant genes other than the RPF-SK1 gene as a parent line, It can be produced relatively easily.
- the selection of spinach plants having the RPF-SK1 gene from the segregating population is carried out, for example, by inoculating the leaves of test plant individuals with Pfs of each race and examining downy mildew resistance. can be done. From the segregating population, plant individuals having resistance to at least all races represented by downy mildew races Pfs1 to Pfs19, UA1014 type, Be2105B type and PV2144 type are selected as individuals having the RPF-SK1 gene.
- Selection of individuals having the RPF-SK1 gene in the selection process can be performed using the RPF-SK1 locus of spinach plants and DNA markers in the vicinity thereof.
- the DNA markers and specific methods to be used can be carried out in the same manner as in the aforementioned screening method according to the present invention.
- the other downy mildew-resistant DNA markers used for selecting individuals having the RPF-SK1 gene are selected.
- a DNA marker capable of identifying the presence or absence of the sex gene is used.
- an SNP or the like present in the gene locus of the other downy mildew-resistant gene and its vicinity can be used.
- the number of repetitions of self-breeding is not particularly limited as long as it is one or more times, and may be 2 to 3 times, or may be 3 times or more.
- This tray was raised in a glass greenhouse for 2 weeks and inoculated with a Pfs spore suspension (5 ⁇ 10 4 /mL) by spraying at the true leaf 2-leaf stage. Immediately after inoculation, the tray was covered with a plastic cover and maintained under the conditions of a temperature of 15° C., a humidity of 100%, and a day length of 12 hours. 7 to 10 days after inoculation, after fully confirming the onset of the disease in the control group, resistance and susceptibility were determined for each individual. Strains in which hyphae and spores appeared on cotyledons or true leaves and the disease was observed were regarded as susceptible, and strains in which neither cotyledons nor true leaves were observed to develop the disease were regarded as resistant.
- Downy mildew races were obtained in Japan from The University of Arkansas (Dr. Jim Correll) and Naktuinbouw. Also, for race discrimination of downy mildew, a known differential set of spinach cultivars showing different resistance patterns to each race can be used as discriminant cultivars. Differential cultivars used to determine downy mildew resistance of each race are available from USDA (United States Department of Agriculture) and Naktuinbouw (Non-Patent Document 4).
- Tables 2 and 3 show the resistance reported for each discriminated variety.
- "-" is resistant
- “(-)” is moderately resistant
- "+” is susceptible
- “(+)” is downy mildew symptoms and spores. Formation is observed only on cotyledons and not on true leaves, respectively.
- “*" means that resistance results differ depending on individual tests.
- Example 1 From 2015 to 2016, a new downy mildew-resistant line was created using the wild Spinacia tetrandra line RNR140003 obtained from CGN in the Netherlands as a parent line.
- F1BC2 seeds were obtained by backcrossing SDF as a pollen parent with an F1BC1 individual whose resistance was confirmed as a seed parent.
- F1BC3 seeds were obtained by backcrossing SDF as a pollen parent with an F1BC2 individual whose resistance was confirmed as a seed parent.
- the applicant deposited the seeds of the SDF (RNR) line with the National Institute of Technology and Evaluation Patent Organism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). Identification given by the depositor of the SDF (RNR) strain: SSC-SPI-21-001, accession number is FERM BP-22426 (deposit date: August 13, 2021).
- Example 2 The Viroflay strain that has no resistance to any race is used as a seed parent, and the SDF (RNR) strain produced in Example 1 is used as a pollen parent, and the obtained F1 individuals are crossed with Pfs1, Pfs2, and Pfs3. , Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19, UA1014 type, Be2105B type, and PV2144 type. inoculation test and investigated the resistance to these.
- the genotype of the RPF-SK1 gene of each individual was obtained by using the genomic DNA extracted from each individual as a template and using the forward primer and reverse primer shown in Table 4 at 94°C for 60 seconds for one cycle, followed by 94°C. 30 seconds at 62°C for 30 seconds, 35 cycles at 72°C for 60 seconds, and 1 cycle at 72°C for 60 seconds. I went by.
- the F1 individuals obtained by crossing the Viroflay line and the SDF (RNR) line showed resistance to all races.
- Pfs1 7 individuals were considered to be disease-resistant, but all of these were individuals showing a disease-resistant response, with only a slight degree of disease on the cotyledons (rather low percentage of rating 5-50%).
- RPF-SK1 gene can confer dominant resistance to at least the races represented by Pfs1 to Pfs19, UA1014 type, Be2105B type, and PV2144 type.
- Example 3 F1 seeds obtained by crossing the Viroflay line as a seed parent and the SDF(RNR) line produced in Example 1 as a pollen parent were selfed to prepare an F2 segregating population. This F2 population was subjected to an inoculation test using races represented by Pfs3, Pfs4, Pfs8, Pfs15, and UA1014 types to examine resistance to these. Table 6 shows the results. The genotypes and the like described in the table are the same as in Table 5.
- the downy mildew resistance of the SDF (RNR) line was close to 3:1 for the Pfs3, Pfs4, Pfs8, Pfs15, and UA1014 types. separated. Therefore, the RPF-SK1 gene was assumed to be monodominant. This assumption was also supported by the results of the Chi - square test (p>0.05).
- the F1 strain obtained by crossing the Viroflay strain and the SDF (RNR) strain produced in Example 1 was selfed to prepare an F2 segregating population.
- an inoculation test was performed using the race indicated by the UA1014 type, and the resistance to this was examined. As a result, there were 76 resistant individuals and 20 susceptible individuals. Genetic analysis was performed on these individuals, and regions in the genome were searched so that resistant individuals would be homozygous or heterozygous, and susceptible individuals would be homozygous.
- Individuals subjected to the trait investigation were used as a population for linkage analysis, and genomic DNA was extracted from each individual. Genotyping was performed using the DNA of the population for linkage analysis and the SNP markers designed on the SpinachBase reference genome. Genotyping was performed using the KASP genotyping assay.
- the SNP identified by chr3_1215815 is cytosine
- the SNP identified by chr3_1215855 is thymine
- the SNP identified by chr3_1216014 is cytosine.
- the SNP identified by chr3_1216093 is guanine
- the SNP identified by chr3_1216094 is guanine
- the SNP identified by chr3_1216095 is adenine
- the SNP identified by chr3_1216288 is adenine
- the SNP identified by chr3_1216291 is The SNP found was guanine.
- the RPF-SK1 gene was different in genotype from the reference genome and the known WOLF (RPF) gene, and the RPF-SK1 gene was a novel downy mildew-resistant gene.
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Abstract
Description
本願は、2021年11月1日に日本に出願された特願2021-178897号に基づき優先権を主張し、その内容をここに援用する。
[1] 少なくとも一方のアレルにおいて、べと病抵抗性RPF-SK1遺伝子を有しており、
前記RPF-SK1遺伝子は、
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである、
べと病抵抗性ホウレンソウ植物。
[2] 前記RPF-SK1遺伝子が、ホモ接合性又はヘテロ接合性である、前記[1]のべと病抵抗性ホウレンソウ植物。
[3] 少なくとも、べと病のレースPfs1、Pfs2、Pfs3、Pfs4、Pfs5、Pfs6、Pfs7、Pfs8、Pfs9、Pfs10、Pfs11、Pfs12、Pfs13、Pfs14、Pfs15、Pfs16、Pfs17、Pfs18、Pfs19、及びUA1014タイプに抵抗性である、及びUA1014タイプが示すレースに抵抗性である、前記[1]又は[2]のべと病抵抗性ホウレンソウ植物。
[4] 少なくとも、べと病のBe2105Bタイプ及びPV2144タイプに抵抗性である、前記[1]~[3]のいずれかのべと病抵抗性ホウレンソウ植物。
[5] 前記べと病抵抗性ホウレンソウ植物が、スピナシア・テトランドラと栽培種ホウレンソウの種間雑種植物に由来するものである、前記[1]~[4]のいずれかのべと病抵抗性ホウレンソウ植物。
[6] 受託番号FERM BP-22426で特定される植物由来のべと病抵抗性を有する、前記[1]~[5]のいずれかのべと病抵抗性ホウレンソウ植物。
[7] 前記RPF-SK1遺伝子以外のべと病抵抗性遺伝子を少なくとも1種以上有する、前記[1]~[6]のいずれかのべと病抵抗性ホウレンソウ植物。
[8] 受託番号FERM BP-22426で特定されるべと病抵抗性ホウレンソウ植物、前記べと病抵抗性ホウレンソウ植物を親系統として得られた雑種植物、又はそれらの後代である、べと病抵抗性ホウレンソウ植物。
[9] 被験ホウレンソウ植物のchr3_1215815、chr3_1215855、chr3_1216014、chr3_1216093、chr3_1216094、chr3_1216095、chr3_1216288、及びchr3_1216291からなる群より選択される1種以上の遺伝子型を調べ、少なくとも一方のアレルにおいて、
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである場合に、
当該被験ホウレンソウ植物がべと病抵抗性である可能性が高いと予測する、ホウレンソウ植物のべと病抵抗性の予測方法。
[10] 被験ホウレンソウ植物のchr3_1215815、chr3_1215855、chr3_1216014、chr3_1216093、chr3_1216094、chr3_1216095、chr3_1216288、及びchr3_1216291からなる群より選択される1種以上の遺伝子型を調べ、少なくとも一方のアレルにおいて、
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである場合に、
当該被験ホウレンソウ植物を、べと病抵抗性ホウレンソウ植物として選抜する、べと病抵抗性ホウレンソウ植物のスクリーニング方法。
[11] RPF-SK1遺伝子を有するホウレンソウ植物と任意のホウレンソウ植物とを交雑する第1交雑工程と、
前記第1交雑工程により得られたF1個体に対して、自殖、戻し交雑、又は前記第1交雑工程において用いた親系統とは異なるホウレンソウ植物との種間交雑又は種内交雑を行い、分離集団を得る第2交雑工程と、
前記分離集団から、RPF-SK1遺伝子を有するホウレンソウ植物を選抜する選抜工程と、
を有する、べと病抵抗性ホウレンソウ植物の製造方法。
[12] 前記任意のホウレンソウ植物、又は前記第2交雑工程における種間交雑又は種内交雑で用いる親系統が、前記RPF-SK1遺伝子以外のべと病抵抗性遺伝子を少なくとも1種以上有するホウレンソウ植物である、前記[11]のべと病抵抗性ホウレンソウ植物の製造方法。
[13] 前記[1]~[8]のいずれかのべと病抵抗性ホウレンソウ植物の植物体の一部。
[14] 前記[1]~[8]のいずれかのべと病抵抗性ホウレンソウ植物の葉。
[15] 前記[1]~[8]のいずれかのべと病抵抗性ホウレンソウ植物の種子。
[16] 被験ホウレンソウ植物のchr3_1215815~chr3_1216291を含む領域をPCR増幅するためのフォワードプライマー及びリバースプライマーを含む、
RPF-SK1遺伝子を有するホウレンソウ植物を選抜するために用いられる、キット。
また、本発明に係るホウレンソウ植物を親系統として利用することにより、広範なレースに対して抵抗性を示す新規ホウレンソウ系統を育成することもできる。
よって、とあるホウレンソウ植物の第X染色体は、当該ホウレンソウ植物のゲノムDNAの塩基配列とSpinachBaseのリファレンスゲノムの塩基配列を、最もホモロジー(配列同一性)が高くなるようにアラインメントすることにより決定したホウレンソウ植物の第X染色体として示すことができる。
本発明に係るべと病抵抗性ホウレンソウ植物は、野生種ホウレンソウであるスピナシア・テトランドラ系統RNR140003のRPF遺伝子座に座上するべと病抵抗性遺伝子であるRPF-SK1遺伝子を有している、スピナシア・テトランドラ以外のホウレンソウ植物である。RPF-SK1遺伝子は、ホウレンソウ植物のRPF遺伝子のうち、下記のいずれかのSNPを有する遺伝子である。
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである。
また、本発明に係るべと病抵抗性ホウレンソウ植物の組織から採取された細胞も有用である。当該細胞は、ホウレンソウ植物のゲノムDNAを有しており、かつRPF-SK1遺伝子を有する植物細胞である。RPF-SK1遺伝子を有するホウレンソウ植物細胞は、単一細胞であってもよく、複数の細胞からなる細胞塊であってもよい。RPF-SK1遺伝子を有するホウレンソウ植物細胞としては、ホウレンソウ植物のいずれの組織から採取されたものであってもよく、異なる組織から採取された細胞を混合した細胞混合物であってもよい。当該組織としては、前記で列挙されたものが挙げられる。中でも、葉身又は葉柄の組織由来の単一細胞が好ましい。
本発明に係るべと病抵抗性ホウレンソウ植物のスクリーニング方法は、被験ホウレンソウ植物がRPF-SK1遺伝子を有するか否かを調べ、RPF-SK1遺伝子を有すると判断された場合に、当該被験ホウレンソウ植物をべと病抵抗性ホウレンソウ植物として選抜する。本発明に係るべと病抵抗性ホウレンソウ植物のスクリーニング方法では、被験ホウレンソウ植物がRPF-SK1遺伝子を有するか否かを、RPF-SK1遺伝子座又はその近傍のDNAマーカーや、これらのDNAマーカーと強く連鎖するDNAマーカーの遺伝子型に基づいて判断する。少なくとも一方のアレルにおいて、RPF-SK1遺伝子座又はその近傍のDNAマーカーやこれらのDNAマーカーと強く連鎖するDNAマーカーの遺伝子型が、RNR140003株等のようなRPF-SK1遺伝子を有し、べと病抵抗性を示すスピナシア・テトランドラと同じ遺伝子型の場合には、当該被験ホウレンソウ植物をべと病抵抗性ホウレンソウ植物として選抜する。
本発明に係るホウレンソウ植物のべと病抵抗性の予測方法は、被験ホウレンソウ植物がRPF-SK1遺伝子を有するか否かを調べ、RPF-SK1遺伝子を有すると判断された場合に、当該被験ホウレンソウ植物がべと病抵抗性である可能性が高いと予測する。本発明に係るホウレンソウ植物のべと病抵抗性の予測方法では、被験ホウレンソウ植物がRPF-SK1遺伝子を有するか否かを、RPF-SK1遺伝子座又はその近傍のDNAマーカーや、これらのDNAマーカーと強く連鎖するDNAマーカーの遺伝子型に基づいて判断する。少なくとも一方のアレルにおいて、RPF-SK1遺伝子座又はその近傍のDNAマーカーやこれらのDNAマーカーと強く連鎖するDNAマーカーの遺伝子型が、RNR140003株等のようなRPF-SK1遺伝子を有し、べと病抵抗性を示すスピナシア・テトランドラと同じ遺伝子型の場合には、当該被験ホウレンソウ植物がRPF-SK1遺伝子を有すると判断し、RNR140003株等のようなRPF-SK1遺伝子を有し、べと病抵抗性を示すスピナシア・テトランドラと異なる遺伝子型の場合には、当該被験ホウレンソウ植物がRPF-SK1遺伝子を有さないと判断する。
RPF-SK1遺伝子を有するホウレンソウ植物を材料として交雑を行うことによって、RPF-SK1遺伝子を有していないホウレンソウ植物にRPF-SK1遺伝子に由来するべと病抵抗性を導入して新規のべと病抵抗性ホウレンソウ植物を製造することができる。単一優性の抵抗性遺伝子であるRPF-SK1遺伝子は、任意の系統へ導入することが容易であり、ホウレンソウの品種育成を加速させることができる。例えば、RPF-SK1遺伝子をホモ接合で有するホウレンソウ植物を親系統として用いることにより、より広範囲なレースに抵抗性を有する品種の育成が容易になる。また、RPF-SK1遺伝子は、RPF-SK1遺伝子座とは異なる遺伝子座にあるその他の単一優性抵抗性対立遺伝子と同一のハプロタイプに併せ持つことが可能である。
ホウレンソウ植物に対するPfsの接種試験は、下記の方法で行った。
ホウレンソウ種子を培土「スーパーミックスA」(サカタのタネ社製)を詰めたトレイに播種し、培土「メトロミックス350」(ハイポネックスジャパン社製)を用いて覆土をした。播種する際、各トレイに、接種するレースに対して罹病性を示す系統の種子を播種し、発病の対照区を設けた。このトレイをガラス温室内で2週間育苗し、本葉2葉期に、Pfsの胞子懸濁液(5 × 104/mL)を噴霧接種した。接種後、ただちにトレイをプラスチックカバーで覆い、温度15℃、湿度100%、12時間日長の条件下で管理した。
接種から7~10日後、対照区の発病を十分に確認した後に、個体ごとに抵抗性・罹病性の判定を行った。子葉又は本葉に菌糸及び胞子が出現して発病が認められた株を罹病性とし、子葉及び本葉ともに発病が認められなかった株を抵抗性とした。
2015年~2016年に、オランダのCGNより入手した野生種スピナシア・テトランドラ系統RNR140003を親系統として、新規なべと病抵抗性系統を作出した。
次いで、このスピナシア・テトランドラ系統RNR140003の個体と、優良親系統であるスピナシア・オレラシア L系統SDFを交配し、F1種子を得た。なお、優良親系統とは、栽培種として好適な形質を発現する系統を作出するための親系統として好適な系統を意味する。
得られたF1種子18粒を播種し、発芽した個体に対してPfs10を接種した。この結果、全個体が抵抗性を示した。抵抗性が確認された個体を種子親として、SDFを花粉親として戻し交配し、F1BC1種子を得た。得られたF1BC1種子を播種し、生育した個体に対し、SDFが抵抗性を示さないPfs10を接種したところ、抵抗性個体(R)と罹病性個体(抵抗性を示さない個体)(S)は、R:S=7:4に分離した。
F1BC1個体のうちの抵抗性が確認された個体を種子親として、SDFを花粉親として戻し交配し、F1BC2種子を得た。得られたF1BC2種子を播種し、生育した個体に対し、Pfs10を接種したところ、R:S=15:28に分離した。
F1BC2個体のうちの抵抗性が確認された個体を種子親として、SDFを花粉親として戻し交配し、F1BC3種子を得た。得られたF1BC3種子を播種し、生育した個体に対し、Pfs10を接種したところ、R:S=28:38に分離した。
F1BC3個体のうちの抵抗性が確認された個体を自殖して、F1BC3S1種子を得た。得られたF1BC3S1種子を播種し、生育した個体に対し、Pfs10を接種したところ、R:S=18:2に分離した。
F1BC3S1個体のうちの抵抗性が確認された個体を自殖し、F1BC3S2種子を得た。得られたF1BC3S2種子を播種し、生育した個体に対し、Pfs10を接種したところ、R:S=26:0であり、抵抗性型に固定していた。Pfs10に対する抵抗性が確認された全個体に、Pfs12を接種したところ、R:S=23:0であり、抵抗性型に固定していた。つまり、F1BC3S2個体は、全て、Pfs10とPfs12の両方に対する抵抗性を有していた。この系統を集団採種し、SDF(RNR)系統と命名した。
いずれのレースにも抵抗性を有さないViroflay系統を種子親、実施例1で作出したSDF(RNR)系統を花粉親として交雑を行い、得られたF1個体に対して、Pfs1、Pfs2、Pfs3、Pfs4、Pfs5、Pfs6、Pfs7、Pfs8、Pfs9、Pfs10、Pfs11、Pfs12、Pfs13、Pfs14、Pfs15、Pfs16、Pfs17、Pfs18、Pfs19、UA1014タイプ、Be2105Bタイプ、及びPV2144タイプが示すレースを用いた接種試験を行い、これらに対する抵抗性を調べた。なお、ホウレンソウは雌雄異株の作物であるが、その同一個体に雌ずいと雄ずいの両方の器官を生じる間性を示す系統も存在する。Viroflayを種子親にしたF1採種においては、Viroflayの自殖種子が含まれてしまうことがある。
Viroflay系統を種子親、実施例1で作出したSDF(RNR)系統を花粉親として交雑して得られたF1種子を自殖し、F2分離集団を作製した。このF2集団に対して、Pfs3、Pfs4、Pfs8、Pfs15、及びUA1014タイプが示すレースを用いた接種試験を行い、これらに対する抵抗性を調べた。結果を表6に示す。表中に記載した遺伝子型等は表5と同じである。
RPF-SK1遺伝子の染色体の座上領域を特定するための遺伝解析及び遺伝子マーカー作製を行った。
得られたF2分離集団96個体について、UA1014タイプが示すレースを用いた接種試験を行い、これに対する抵抗性を調べた。この結果、抵抗性個体が76個体、罹病性個体が20個体であった。
これらの個体の遺伝解析を行い、抵抗性個体では抵抗性ホモ型又はヘテロ型となり、罹病性個体では罹病性ホモ型となるような領域を、ゲノム中から探索した。
形質調査を行った個体を連鎖解析用集団とし、各個体からゲノムDNAを抽出した。連鎖解析用集団のDNAと、SpinachBaseのリファレンスゲノム上に設計したSNPマーカーを用いて、ジェノタイピングを行った。ジェノタイピングは、KASPジェノタイピングアッセイを用いて行った。
Claims (16)
- 少なくとも一方のアレルにおいて、べと病抵抗性RPF-SK1遺伝子を有しており、
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである、
べと病抵抗性ホウレンソウ植物。 - 前記RPF-SK1遺伝子が、ホモ接合性又はヘテロ接合性である、請求項1に記載のべと病抵抗性ホウレンソウ植物。
- 少なくとも、べと病のレースPfs1、Pfs2、Pfs3、Pfs4、Pfs5、Pfs6、Pfs7、Pfs8、Pfs9、Pfs10、Pfs11、Pfs12、Pfs13、Pfs14、Pfs15、Pfs16、Pfs17、Pfs18、Pfs19、及びUA1014タイプが示すレースに抵抗性である、請求項1に記載のべと病抵抗性ホウレンソウ植物。
- 少なくとも、べと病のBe2105Bタイプ及びPV2144タイプが示すレースに抵抗性である、請求項1に記載のべと病抵抗性ホウレンソウ植物。
- 前記べと病抵抗性ホウレンソウ植物が、スピナシア・テトランドラと栽培種ホウレンソウの種間雑種植物に由来するものである、請求項1に記載のべと病抵抗性ホウレンソウ植物。
- 受託番号FERM BP-22426で特定される植物由来のべと病抵抗性を有する、請求項1に記載のべと病抵抗性ホウレンソウ植物。
- 前記RPF-SK1遺伝子以外のべと病抵抗性遺伝子を少なくとも1種以上有する、請求項1に記載のべと病抵抗性ホウレンソウ植物。
- 受託番号FERM BP-22426で特定されるべと病抵抗性ホウレンソウ植物、前記べと病抵抗性ホウレンソウ植物を親系統として得られた雑種植物、又はそれらの後代である、べと病抵抗性ホウレンソウ植物。
- 被験ホウレンソウ植物のchr3_1215815、chr3_1215855、chr3_1216014、chr3_1216093、chr3_1216094、chr3_1216095、chr3_1216288、及びchr3_1216291からなる群より選択される1種以上の遺伝子型を調べ、少なくとも一方のアレルにおいて、
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである場合に、
当該被験ホウレンソウ植物がべと病抵抗性である可能性が高いと予測する、ホウレンソウ植物のべと病抵抗性の予測方法。 - 被験ホウレンソウ植物のchr3_1215815、chr3_1215855、chr3_1216014、chr3_1216093、chr3_1216094、chr3_1216095、chr3_1216288、及びchr3_1216291からなる群より選択される1種以上の遺伝子型を調べ、少なくとも一方のアレルにおいて、
(a)chr3_1215815で特定されるSNPがシトシンである、
(b)chr3_1215855で特定されるSNPがチミンである、
(c)chr3_1216014で特定されるSNPがシトシンである、
(d)chr3_1216093で特定されるSNPがグアニンであり、chr3_1216094で特定されるSNPがグアニンであり、かつchr3_1216095で特定されるSNPがアデニンである、
(e)chr3_1216288で特定されるSNPがアデニンである、又は
(f)chr3_1216291で特定されるSNPがグアニンである場合に、
当該被験ホウレンソウ植物を、べと病抵抗性ホウレンソウ植物として選抜する、べと病抵抗性ホウレンソウ植物のスクリーニング方法。 - RPF-SK1遺伝子を有するホウレンソウ植物と任意のホウレンソウ植物とを交雑する第1交雑工程と、
前記第1交雑工程により得られたF1個体に対して、自殖、戻し交雑、又は前記第1交雑工程において用いた親系統とは異なるホウレンソウ植物との種間交雑又は種内交雑を行い、分離集団を得る第2交雑工程と、
前記分離集団から、RPF-SK1遺伝子を有するホウレンソウ植物を選抜する選抜工程と、
を有する、べと病抵抗性ホウレンソウ植物の製造方法。 - 前記任意のホウレンソウ植物、又は前記第2交雑工程における種間交雑又は種内交雑で用いる親系統が、前記RPF-SK1遺伝子以外のべと病抵抗性遺伝子を少なくとも1種以上有するホウレンソウ植物である、請求項11に記載のべと病抵抗性ホウレンソウ植物の製造方法。
- 請求項1~8のいずれか一項に記載のべと病抵抗性ホウレンソウ植物の植物体の一部。
- 請求項1~8のいずれか一項に記載のべと病抵抗性ホウレンソウ植物の葉。
- 請求項1~8のいずれか一項に記載のべと病抵抗性ホウレンソウ植物の種子。
- 被験ホウレンソウ植物のchr3_1215815~chr3_1216291を含む領域をPCR増幅するためのフォワードプライマー及びリバースプライマーを含む、
RPF-SK1遺伝子を有するホウレンソウ植物を選抜するために用いられる、キット。
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- 2022-11-01 CN CN202280068472.3A patent/CN118103506A/zh active Pending
- 2022-11-01 WO PCT/JP2022/040890 patent/WO2023074911A1/ja active Application Filing
- 2022-11-01 JP JP2023556710A patent/JPWO2023074911A1/ja active Pending
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KR20240099198A (ko) | 2024-06-28 |
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