WO2016148279A1 - Powdery mildew resistance-associated markers of fragaria genus plants and use thereof - Google Patents

Powdery mildew resistance-associated markers of fragaria genus plants and use thereof Download PDF

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WO2016148279A1
WO2016148279A1 PCT/JP2016/058711 JP2016058711W WO2016148279A1 WO 2016148279 A1 WO2016148279 A1 WO 2016148279A1 JP 2016058711 W JP2016058711 W JP 2016058711W WO 2016148279 A1 WO2016148279 A1 WO 2016148279A1
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powdery mildew
strawberry
marker
genus plant
mildew resistance
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PCT/JP2016/058711
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French (fr)
Japanese (ja)
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弘明 小石原
宏征 榎
村松　正善
西村　哲
進 由比
正憲 本城
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トヨタ自動車株式会社
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Priority claimed from JP2016042028A external-priority patent/JP6566480B2/en
Application filed by トヨタ自動車株式会社 filed Critical トヨタ自動車株式会社
Priority to US15/558,663 priority Critical patent/US10724093B2/en
Priority to CN201680010637.6A priority patent/CN107250357B/en
Publication of WO2016148279A1 publication Critical patent/WO2016148279A1/en
Priority to US16/704,570 priority patent/US11384395B2/en

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • 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
    • 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

Definitions

  • the present invention relates to a powdery mildew resistance-related marker capable of selecting a strawberry genus plant line exhibiting resistance to powdery mildew and a method for using the same.
  • DNA markers also referred to as genetic markers or genetic markers
  • development of DNA markers is progressing not only in model plants such as Arabidopsis thaliana and rice, but also in various practical plants, which is very useful in breeding.
  • Non-Patent Document 1 reports that there are at least two races on strawberry powdery mildew in Japan. Non-patent document 1 suggests that the powdery mildew resistance of strawberries is governed by at least one main gene from the results of investigating the susceptibility and resistance to powdery mildew. However, Non-Patent Document 1 does not disclose or suggest a DNA marker related to powdery mildew resistance in strawberry.
  • Non-Patent Document 2 discloses that a linkage map was created using hybrid individuals of the strawberry cultivar “Toyooka” ⁇ “Takarakosei” and a powdery mildew resistant DNA marker was selected.
  • 29 linkage groups total 109 markers, total length 1451.7 cM
  • 21 linkage groups total 88 markers, total length 1205.7 cM
  • the LOD value is about 1.22 in a linkage group considered promising.
  • Non-Patent Document 3 resistance to strawberry powdery mildew is due to the accumulation of multiple resistance genes, strawberry powdery mildew susceptible cultivar "Sachinoka” and F. ⁇ virginiana (prototype) having resistance are used. It is disclosed that a linkage map of 30 linkage groups with a total length of 1360 cM by 137 DNA markers was prepared. According to Non-Patent Document 3, QTLs are positioned at three locations by the QTL analysis using strawberry powdery mildew test results and a linkage map.
  • the present invention has developed a large number of DNA markers for polyploidy and complicated genome structure of Strawberry genus plants, and using these many DNA markers, the powdery mildew resistance is highly accurate. It is an object of the present invention to provide a powdery mildew resistance-related marker that can be determined by the method and a method for using the marker.
  • the present inventors prepared a number of markers in Strawberry genus plants, and developed the resistance to powdery mildew by analyzing the expression of the traits in the hybrid progeny line and the linkage between the markers. As a result, the present inventors have completed the present invention.
  • the present invention includes the following.
  • a strawberry genus plant powdery mildew resistance-related marker comprising a continuous nucleic acid region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 19 in the chromosome of a strawberry genus plant.
  • the nucleic acid region is located in a region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 7 in the chromosome of the strawberry genus plant.
  • Powdery mildew resistance related marker (4) a step of extracting a chromosome of a progeny plant in which at least one parent is a strawberry genus plant and / or a chromosome of the parent strawberry genus plant, and any of the above (1) to (3) in the chromosome obtained above
  • a method for producing a strawberry genus plant line having improved powdery mildew resistance comprising the step of determining the presence / absence of a marker associated with resistance to powdery mildew genus plant according to claim 1.
  • determining step presence / absence of the strawberry genus plant powdery mildew resistance-related marker by a nucleic acid amplification reaction using a primer that specifically amplifies the strawberry genus plant powdery mildew resistance-related marker.
  • (6) The method for producing a strawberry genus plant line according to (4), wherein in the determining step, a DNA chip comprising a probe corresponding to the strawberry genus plant powdery mildew resistance-related marker is used.
  • the present invention it is possible to provide a novel strawberry genus plant powdery mildew resistance-related marker linked to powdery mildew resistance among traits in strawberry plants.
  • the strawberry genus plant powdery mildew resistance-related marker according to the present invention powdery mildew resistance in a hybrid strain of strawberry genus plants can be tested.
  • the Strawberry genus plant line which has the characteristic which powdery mildew resistance improved can be identified at very low cost.
  • a characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there.
  • a characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there.
  • FIG. 5 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB535110 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and ⁇ f”.
  • FIG. 5 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB535110 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and ⁇ f”.
  • It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (B group) of hybridization of "Miyazaki Natsuka Haruka” and "Ookimi".
  • FIG. 6 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB522828 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and ⁇ f”.
  • FIG. 6 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB522828 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and ⁇ f”.
  • FIG. 6 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB522828 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and ⁇ f”.
  • It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (B group) of a hybrid between "Miyazaki Natsuka Haruka” and "Ookimi".
  • the strawberry genus plant powdery mildew resistance-related marker according to the present invention and a method of using the marker, particularly the strawberry genus plant strain-related production method using the strawberry genus plant powdery mildew resistance-related marker will be described.
  • the strawberry genus plant powdery mildew resistance-related marker according to the present invention is a specific region present on the chromosome of a strawberry genus plant, and has a function of distinguishing the trait of strawberry genus plant powdery mildew resistance. That is, in the progeny line obtained using a known strawberry genus plant, by confirming the presence / absence of the strawberry genus plant powdery mildew resistance-related marker, whether the line has resistance to powdery mildew Can be determined.
  • strawberry powdery mildew is a disease caused by infection of Sphaerotheca aphanis (Podosphaera aphanis) as described in Ann. Phytopathol. Soc. Jpn. 64: 121-l24, l998. It means the disease that the group is formed.
  • strawberry powdery mildew particularly means “Toyonoka”, “Nyoho”, “Rei”, “Himiko”, “Treasure early life”, “Danner”, “Sachitama”, Among the 9 varieties of “Haruno” and “Fukuha”, strains that show no pathogenicity in “Toyonoka” and “Haruno”, but are pathogenic in the other 7 varieties Report 63, No. 3, page 226).
  • Strawberry plant powdery mildew resistance-related marker means a marker linked to a trait that is highly resistant to powdery mildew. For example, in a specific strawberry genus plant, if a strawberry genus plant powdery mildew resistance-related marker exists, it can be determined that the variety has high powdery mildew resistance. In particular, the strawberry genus plant powdery mildew resistance-related marker is considered to be a region linked to a causative gene (group) of traits such as powdery mildew resistance of strawberry plants.
  • the strawberry genus plant is meant to include all plants belonging to the genus Fragaria L. That is, examples of the Strawberry genus plant include hybrids such as Dutch strawberry (Fragaria ⁇ ananassa) which is a general cultivated strawberry.
  • Strawberry genus plants include F. virginiana and F. chiloensis, Ezoxa strawberry (F. iinumae), white snake strawberry (F. nipponica), F. nilgerrensis, which are ancestors of cultivated strawberries.
  • wild species such as F.colanubicola, F. bucharica, F. daltoniana, F. orientalis, F. corimbosa, F. moschata and F. iturupensis.
  • a strawberry genus plant it is the meaning also including the known varieties and strains in cultivated strawberries (F. F x ananassa).
  • cultivated strawberries F. F x ananassa
  • Known varieties / lines of cultivated strawberries are not particularly limited, and include all varieties / lines that can be used in Japan, varieties / lines that are used outside of Japan, and the like.
  • cultivated strawberry cultivated varieties in Japan are not particularly limited, but toyoka, Santigo, pure berry, Nyoho, Peace Toro, Linda Mall, Tochiotome, Ice Toro, Tochinomine, Akihime, Beni Hoppe, Tochi Hime, Sachinoka, Keikise, Sagahonoka, Iberry, Karenberry, Red Pearl, Otome Satsuma, Fukuoka S6 (Amaou), Ninohime, Hinone, and Hyocho Precocious.
  • group should just be a strawberry genus plant in which any one of a mother book and a father's book mentioned above, the system
  • the progeny line may be obtained by so-called backcrossing.
  • the new variety is a variety obtained from at least one parent having a strawberry powdery mildew resistance.
  • the strawberry genus plant powdery mildew resistance-related marker according to the present invention is a gene linkage comprising 8,218 markers obtained from the strawberry cultivar “Miyazaki Natsuka Haruka” and 8,039 markers obtained from the strawberry line “08 and -f”. It was newly identified by QTL (Quantitative Trait Loci) analysis using a map and strawberry powdery mildew resistance data. Genetic analysis software QTL Cartographer (Wang S., C. J. Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC) The Composite interval mapping (CIM) method is applied.
  • QTL Quantitative Trait Loci
  • a region where the rod score (LOD score) is a predetermined threshold (for example, 2.5) or more was found in the gene linkage map.
  • the region 1 LOD down from the peak was about 6.8 cM (centiorgan), which was a region included in the first linkage group of “08 and ⁇ f”.
  • Morgan (M) is a unit that relatively indicates the distance between genes on a chromosome, and is a value obtained by setting the cross value as a percentage.
  • 1 cM corresponds to about 400 kb.
  • the 19 kinds of markers shown in Table 1 are included in this order in this 6.8 cM region.
  • the marker name is a name given to the marker uniquely acquired in the present invention.
  • the continuous nucleic acid region contained in the 6.8 cM region shown in Table 1 can be used as a marker associated with resistance to powdery mildew plant genus.
  • the nucleic acid region is such that the identity with other regions existing in the chromosome of the strawberry genus plant is 95% or less, preferably 90% or less, more preferably 80% or less, and most preferably 70% or less. This means a region consisting of a simple base sequence. If the identity of the nucleic acid region serving as a marker associated with resistance against powdery mildew plant and other regions is within the above range, the nucleic acid region can be specifically detected according to a standard method.
  • the identity value can be calculated using default parameters using, for example, BLAST.
  • the base length of the nucleic acid region serving as a strawberry genus plant powdery mildew resistance-related marker is at least 8 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and most preferably 30 bases in length. be able to. If the base length of the nucleic acid region serving as a strawberry genus plant powdery mildew resistance-related marker is within the above range, the nucleic acid region can be specifically detected according to a conventional method.
  • the strawberry genus plant powdery mildew resistance-related marker is a region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 7 among 19 types of markers contained in the 6.8 cM region. Is preferably selected from. This is because the peak exists in a region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 7.
  • the strawberry genus plant powdery mildew resistance-related marker may be a nucleic acid region containing one type of marker selected from the 19 types of markers shown in Table 1 above.
  • a strawberry genus plant powdery mildew resistance-related marker it is preferable to use a nucleic acid region containing a marker (IB535110) consisting of the base sequence shown in SEQ ID NO: 1 closest to the peak position.
  • the base sequence of the nucleic acid region containing the marker can be specified by an adjacent sequence acquisition method such as inverse PCR using a primer designed based on the base sequence of the marker.
  • a plurality of regions can be used as a marker associated with resistance to powdery mildew in the strawberry genus plant from the nucleic acid region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 19 in the chromosome of the strawberry genus plant.
  • the above 19 kinds of markers themselves can be used as the strawberry genus plant powdery mildew resistance-related marker. That is, one or a plurality of regions selected from the 19 regions consisting of the nucleotide sequences of SEQ ID NOS: 1 to 19 can be used as a strawberry genus plant powdery mildew resistance-related marker.
  • a marker IB535110 comprising the base sequence shown in SEQ ID NO: 1 closest to the peak position.
  • a region sandwiched between a marker consisting of the base sequence of SEQ ID NO: 2 (IB522828) and a marker consisting of the base sequence of SEQ ID NO: 3 (IB559302) can be used as a strawberry plant powdery mildew resistance-related marker.
  • the strawberry genus plant powdery mildew resistance-related marker is obtained from 8,218 markers obtained from the strawberry cultivar “Miyazaki Natsuka Haruka” and 8,039 markers obtained from the strawberry line “08 and -f”. Identified. Here, these 8,218 and 8,039 markers will be described.
  • a DNA microarray to which the methods disclosed in JP 2011-120558 A and International Publication 2011/074510 are applied can be used.
  • the design method of the probe used for the DNA microarray first extracts genomic DNA from “Miyazaki Natsuki Haruka” or “08 and -f” (step 1a). Next, the extracted genomic DNA is digested with one or more restriction enzymes (step 1b).
  • genomic DNA is digested using two types of restriction enzymes, restriction enzyme A and restriction enzyme B, in this order.
  • restriction enzyme is not particularly limited, and for example, PstI, EcoRI, HindIII, BstNI, HpaII, HaeIII and the like can be used.
  • the restriction enzyme can be appropriately selected in consideration of the appearance frequency of the recognition sequence so that a genomic DNA fragment having a length of 20 to 10,000 bases is obtained when the genomic DNA is completely digested.
  • a plurality of restriction enzymes it is preferable that the genomic DNA fragment after using all restriction enzymes has a length of 200 to 6000 bases.
  • the order of restriction enzymes to be used for the treatment is not particularly limited, and when the processing conditions (solution composition, temperature, etc.) are common, a plurality of restriction enzymes are used in the same reaction system. May be used in That is, in the example shown in FIG.
  • restriction enzyme A and restriction enzyme B are used in this order to digest genomic DNA, but restriction enzyme A and restriction enzyme B are simultaneously used in the same reaction system.
  • Genomic DNA may be digested, or genomic DNA may be digested using restriction enzyme B and restriction enzyme A in this order. Further, the number of restriction enzymes used may be 3 or more.
  • a plurality of adapters corresponding to each restriction enzyme can be prepared and used. That is, a plurality of adapters having a single strand complementary to each of a plurality of types of protruding ends generated when genomic DNA is digested with a plurality of restriction enzymes can be used.
  • the plurality of adapters corresponding to the plurality of restriction enzymes may have a common primer binding sequence so that a common primer can hybridize, or different primers can hybridize with each other. May have different primer binding sequences.
  • the adapter when digesting genomic DNA using a plurality of restriction enzymes, may be one restriction enzyme selected from the plurality of restriction enzymes used or a part of the restriction enzymes used. A corresponding adapter can also be prepared and used.
  • a genomic DNA fragment with adapters added to both ends is amplified (step 1d).
  • the genomic DNA fragment can be amplified by using a primer that can hybridize to the primer binding sequence.
  • a genomic DNA fragment to which an adapter has been added can be cloned into a vector using the adapter sequence, and the genomic DNA fragment can be amplified using a primer that can hybridize to a predetermined region in the vector.
  • PCR can be used as an example of the amplification reaction of a genomic DNA fragment using a primer.
  • genomic DNA fragments obtained by treatment with multiple restriction enzymes An adapter will be connected to all of these.
  • all the genomic DNA fragments obtained can be amplified by performing a nucleic acid amplification reaction using the primer binding sequence contained in the adapter.
  • genomic DNA is digested using a plurality of restriction enzymes, and one restriction enzyme selected from the plurality of restriction enzymes used or an adapter corresponding to a part of the restriction enzymes used is used.
  • one restriction enzyme selected from the plurality of restriction enzymes used or an adapter corresponding to a part of the restriction enzymes used is used.
  • the base sequence of the amplified genomic DNA fragment is determined (step 1e), and one or more regions having a base length shorter than the genomic DNA fragment and covering at least a part of the genomic DNA fragment are identified. Then, the specified region or regions are designed as probes in the cultivated strawberry (step 1f).
  • the method for determining the base sequence of the genomic DNA fragment is not particularly limited, and a conventionally known method using a DNA sequencer to which the Sanger method or the like is applied can be used.
  • the region to be designed is, for example, 20 to 100 bases long, preferably 30 to 90 bases long, more preferably 50 to 75 bases long as described above.
  • DNA microarrays can be made. By using the thus prepared DNA microarray, it is possible to identify 8,218 and 8,039 markers including 19 kinds of strawberry plant powdery mildew resistance-related markers shown in SEQ ID NOS: 1 to 19 above. it can.
  • the present inventors have described the DNA microarray described above for 8,218 markers obtained from the strawberry cultivar “Miyazaki Natsuka Haruka”, strawberry lines “08 and -f” and their progeny lines (147 lines). Signal data was acquired using. And genotype data is obtained from the obtained signal data, and based on this genotype data, genetic map creation software AntMap (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm (Breed Sci 56: 371-378) was used to calculate the position information of the marker on the chromosome using the genetic distance calculation formula Kosambi.
  • AntMap Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm (Breed Sci 56: 371-378) was used to calculate the position information of the marker on the chromosome using the genetic distance calculation formula Kosambi.
  • ⁇ Utilization of markers related to resistance to powdery mildew plant genus> By using a strawberry plant powdery mildew resistance-related marker, it is possible to determine whether a strawberry genus plant (for example, a progeny strain) whose powdery mildew resistance is unknown is a strain exhibiting powdery mildew resistance. it can.
  • using a strawberry genus plant powdery mildew resistance-related marker means a form using a method of specifically amplifying a nucleic acid fragment containing the marker, a form using a DNA microarray having a probe corresponding to the marker It means to include.
  • the method of specifically amplifying a nucleic acid fragment containing a strawberry genus plant powdery mildew resistance-related marker means using a so-called nucleic acid amplification method.
  • the nucleic acid amplification method include a method using a primer designed to specifically amplify a target nucleic acid fragment, and a method of specifically amplifying a target nucleic acid fragment without using a primer.
  • a primer that specifically amplifies a target nucleic acid fragment means an oligonucleotide that can amplify a nucleic acid fragment containing a strawberry genus plant powdery mildew resistance-related marker as defined above by a nucleic acid amplification method.
  • the nucleic acid amplification method using a primer is not particularly limited, and any method may be used as long as it amplifies a nucleic acid fragment typified by a PCR (Polymerase Chain Reaction) method.
  • RCA RollingRCircle Amplification
  • CPT Cycling Probe Technology
  • ICAN Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids
  • LAMP Loop-Mediated Isothermal Amplificaton of
  • SDA Strand Displacement Amplification
  • NASBA Nucleic acid Sequence-based Amplification method
  • TMA Transcription mediated amplification method
  • nucleic acid amplification reactions for example, when using the PCR method, a pair of primers is designed so as to sandwich a strawberry plant powdery mildew resistance-related marker in the chromosome of the strawberry plant.
  • a pair of primers is designed so as to sandwich a strawberry genus plant powdery mildew resistance-related marker in the chromosome of the strawberry plant.
  • four kinds of primers are designed so as to sandwich a strawberry genus plant powdery mildew resistance-related marker in the chromosome of the strawberry genus plant.
  • the nucleic acid amplification method without using a primer is not particularly limited, and examples thereof include an LCR (LigaseigChain Reaction) method.
  • LCR LiigaseigChain Reaction
  • a plurality of oligonucleotides that hybridize to a nucleic acid fragment containing a strawberry genus plant powdery mildew resistance-related marker are designed.
  • nucleic acid amplification method when the strawberry genus plant powdery mildew resistance-related marker is present in the strawberry genus plant to be examined, a nucleic acid fragment containing the marker can be obtained as an amplification product. it can.
  • the desired nucleic acid fragment is amplified by the nucleic acid amplification method using the chromosome extracted from the strawberry plant to be tested as a template, the strawberry genus plant to be tested has powdery mildew resistance. Judgment can be made.
  • a method for observing specific fluorescence by subjecting the solution after amplification reaction to agarose electrophoresis and binding a fluorescent intercalator such as Ethidium Bromide or SYBR Green examples include a method of adding a fluorescent intercalator to a nucleic acid amplification reaction solution to detect fluorescence after the amplification reaction, a method of performing nucleic acid amplification reaction using a fluorescently labeled primer, and detecting fluorescence after the amplification reaction be able to.
  • the base length of the amplified fragment containing the marker varies depending on the principle of the nucleic acid amplification method, etc., for example, 30 to 10,000.
  • the length can be a base length, preferably 50 to 5000 bases, more preferably 70 to 2000 bases.
  • a plurality of strawberry genus plant powdery mildew resistance-related markers may be detected. That is, a plurality of regions selected from the nucleic acid region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 19 in the chromosome of the strawberry genus plant is used as a strawberry genus plant powdery mildew resistance-related marker, Strawberry plant powdery mildew resistance-related markers may be detected. For example, a plurality of regions selected from 19 regions consisting of the base sequences of SEQ ID NOs: 1 to 19 may be used as a strawberry genus plant powdery mildew resistance-related marker, and the plurality of regions may be detected.
  • the region consisting of the nucleotide sequence of SEQ ID NO: 1 (IB535110) and the region consisting of the nucleotide sequence of SEQ ID NO: 2 (IB522828) are used as markers associated with powdery mildew plant resistance, respectively, and each of these regions is amplified by nucleic acid. It can be amplified by the method to confirm the presence or absence of a strawberry genus plant powdery mildew resistance-related marker.
  • a region sandwiched between a region consisting of the base sequence of SEQ ID NO: 2 (IB522828) and a region consisting of the base sequence of SEQ ID NO: 3 (IB559302) is used as a strawberry genus plant powdery mildew resistance related marker, It can be amplified by a nucleic acid amplification method to confirm the presence or absence of a strawberry genus plant powdery mildew resistance-related marker.
  • the probe is a strawberry genus plant powdery mildew resistance-related marker defined as described above.
  • the DNA microarray having the probe may be any microarray using a flat substrate such as glass or silicone as a carrier, a bead array using a microbead as a carrier, or a three-dimensional microarray in which a probe is fixed to the inner wall of a hollow fiber. It may be a type of microarray.
  • strawberry genus plants with unknown powdery mildew resistance phenotypes exhibit a phenotype that is excellent in powdery mildew resistance. It can be judged whether it is a system.
  • the above-mentioned strawberry genus plant powdery mildew resistance-related marker is detected using a conventionally known method, and the powdery mildew resistance of the test strawberry genus plant is detected. You may judge whether it is a strain
  • a so-called FISH (fluorescence in situ hybridization) method using the above-described probe can be applied.
  • genomic DNA is extracted from the strawberry genus plant.
  • This strawberry genus plant is a strawberry genus plant with unknown powdery mildew resistance phenotype and / or a parent strawberry genus plant used when producing a progeny strain, It is a strawberry genus plant for which it is judged whether it has a trait such as excellent disease resistance.
  • the extracted genomic DNA is digested with the restriction enzymes used in preparing the DNA microarray described in the above section ⁇ Identification of Markers in Strawberry Plant> to prepare a plurality of genomic DNA fragments.
  • the obtained genomic DNA fragment is ligated to the adapter used in preparing the DNA microarray.
  • the genomic DNA fragment with adapters added to both ends is amplified using the primers used in preparing the DNA microarray. Thereby, the genomic DNA fragment derived from the strawberry genus plant corresponding to the genomic DNA fragment amplified in step 1d when preparing the DNA microarray can be amplified.
  • a predetermined genomic DNA fragment may be selectively amplified among the genomic DNA fragments to which the adapter is added.
  • a genomic DNA fragment to which a specific adapter is added can be selectively amplified.
  • the adapter is added by adding the adapter only to the genomic DNA fragment having a protruding end corresponding to the predetermined restriction enzyme. Genomic DNA fragments can be selectively amplified. Thus, it can be concentrated by selectively amplifying a predetermined genomic DNA fragment.
  • a label is added to the amplified genomic DNA fragment.
  • Any conventionally known substance may be used as the label.
  • the label for example, fluorescent molecules, dye molecules, radioactive molecules and the like can be used.
  • This step can be omitted by using a nucleotide having a label in the step of amplifying the genomic DNA fragment. This is because the amplified DNA fragment is labeled by amplifying the genomic DNA fragment using a nucleotide having a label in the above step.
  • the genomic DNA fragment having the label is brought into contact with the DNA microarray under predetermined conditions, and the probe immobilized on the DNA microarray and the genomic DNA fragment having the label are hybridized.
  • high stringency conditions for hybridization.
  • Stringency conditions can be adjusted by reaction temperature and salt concentration. That is, higher stringency conditions are achieved at higher temperatures, and higher stringency conditions are achieved at lower salt concentrations. For example, when a probe having a length of 50 to 75 bases is used, higher stringency conditions can be obtained by setting the hybridization conditions to 40 to 44 ° C., 0.2% SDS, and 6 ⁇ SSC.
  • hybridization between the probe and the genomic DNA fragment having a label can be detected based on the label. That is, after the hybridization reaction of the genomic DNA fragment having the above-described label and the probe, the unreacted genomic DNA fragment is washed, and then the label of the genomic DNA fragment specifically hybridized to the probe is observed. .
  • the label is a fluorescent substance
  • the fluorescence wavelength is detected
  • the label is a dye molecule
  • the dye wavelength is detected.
  • an apparatus such as a fluorescence detection apparatus or an image analyzer that is used for normal DNA microarray analysis can be used.
  • the strawberry genus plant powdery mildew resistance-related marker can be determined by a method using a nucleic acid amplification method or a method using a DNA microarray.
  • the strawberry genus plant powdery mildew resistance-related marker is linked to the trait that it is excellent in powdery mildew resistance, so if the strawberry genus plant powdery mildew resistance-related marker exists, It can be judged that it is a variety and strain excellent in powdery mildew resistance.
  • restriction enzyme BstNI NEB, 6 units was added to the treated sample and treated at 60 ° C. for 1 hour.
  • DNA Ligase (NEB, 200 unit) was added, and a ligation reaction was performed at 16 ° C. for 1 hour, then at 55 ° C. for 20 minutes, and then at 37 ° C. for 30 minutes.
  • restriction enzyme BstNI (NEB, 6 units) was added to the treated sample and treated at 60 ° C. for 1 hour.
  • the effect column shows the effect of this QTL on the incidence of powdery mildew (none, 0, 1, 2, 3). That is, when the numerical value of the effect is negative, it acts to reduce the onset of powdery mildew, meaning that the QTL is linked to a trait that improves powdery mildew resistance.
  • progeny seeds of hybrids between “Miyazaki Natsuka Haruka” and “Ookimi” 42 lines: hereinafter referred to as Group B
  • progeny seeds of “Miyazaki Natsuka Haruka” and “09s Eb 45e” 42 lines: subsequent
  • seedlings, planting, and the occurrence of powdery mildew were investigated (FIGS. 5-1 and 5-2).
  • (2) Extraction of genomic DNA Genomic DNA was newly extracted from the strawberry cultivars “Miyazaki Natsuka Haruka”, “08 and -f” and Group A using the Dneasy Plant Mini kit (QIAGEN).
  • FIGS. 6-1 to 6-5) 90.0% to 98.0%.
  • FIGS. 6-1 to 6-5 high array signal values are underlined. From these results, it was considered that by using the markers shown in Table 1, it was possible to select strains with excellent and inferior powdery mildew resistance.
  • FIGS. 10-1 and 10-2 the M lane indicates “Miyazaki Natsuka Haruka”, and the Z lane indicates “08 and ⁇ f”. These results are collectively shown in FIGS. 10-1 and 10-2.
  • the underline indicates the case where the phenotype does not match the PCR base marker result.
  • the coincidence ratio between the band pattern and the phenotype is very high (98.5%), and the noodles are amplified by a nucleic acid amplification method using a primer that specifically amplifies IB535110. It became clear that it was possible to select strains with excellent disease resistance and inferior strains.
  • FIGS. 12-1 and 12-2, and FIGS. 13-1 and 13-2 respectively.
  • the M lane indicates “Miyazaki Natsuka Haruka”
  • the Z lane indicates “08 and ⁇ f”
  • the O lane indicates “Ohmi”.
  • FIGS. 14-1 and 14-2 the underline indicates a case where the phenotype does not match the result of the PCR base marker. As shown in FIGS.
  • the coincidence rate between the band pattern and the phenotype is very high (98.5%), and by using the nucleic acid amplification method using a primer that specifically amplifies IB522828, udon It became clear that it was possible to select strains with excellent disease resistance and inferior strains.

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Abstract

Multiple DNA markers are developed for Fragaria genus plants, and powdery mildew resistance is determined at high accuracy using these multiple DNA markers. A Fragaria genus plant powdery mildew resistance-associated marker comprising a consecutive nucleic acid region flanked by a base sequence represented by SEQ ID NO: 1 and a base sequence represented by SEQ ID NO: 19 in the chromosome of a Fragaria genus plant.

Description

イチゴ属植物のうどんこ病抵抗性関連マーカーとその利用Markers of resistance to powdery mildew in strawberry plants and their utilization
　本発明は、うどんこ病に対する抵抗性を示すイチゴ属植物系統を選抜することができるうどんこ病抵抗性関連マーカー及びその利用方法に関する。 The present invention relates to a powdery mildew resistance-related marker capable of selecting a strawberry genus plant line exhibiting resistance to powdery mildew and a method for using the same.
　DNAマーカー（遺伝マーカーや遺伝子マーカーとも称される）の開発により、植物の品種改良において、有用形質や不良形質の有無を迅速に且つ効率的に判別することが可能となっている。DNAマーカーの開発は、シロイヌナズナやイネといったモデル植物のみならず、様々な実用植物においても進捗しており、品種改良において大いに役立っている。 Development of DNA markers (also referred to as genetic markers or genetic markers) makes it possible to quickly and efficiently determine the presence or absence of useful or defective traits in plant variety improvement. The development of DNA markers is progressing not only in model plants such as Arabidopsis thaliana and rice, but also in various practical plants, which is very useful in breeding.
　非特許文献１には、日本国内におけるイチゴのうどんこ病菌について、少なくとも２レースが存在することが報告されている。また、非特許文献１には、うどんこ病菌に対する感受性及び抵抗性を調査した結果から、イチゴのうどんこ病抵抗性は少なくとも１つの主働遺伝子によって支配されていることが示唆されている。ただし、非特許文献１は、イチゴについてうどんこ病抵抗性に関連するDNAマーカーを開示或いは示唆するものではない。 Non-Patent Document 1 reports that there are at least two races on strawberry powdery mildew in Japan. Non-patent document 1 suggests that the powdery mildew resistance of strawberries is governed by at least one main gene from the results of investigating the susceptibility and resistance to powdery mildew. However, Non-Patent Document 1 does not disclose or suggest a DNA marker related to powdery mildew resistance in strawberry.
　非特許文献２には、イチゴ品種「とよのか」×「宝交早生」の交雑個体を用いて連鎖地図を作成し、うどんこ病抵抗性DNAマーカーを選抜したことが開示されている。非特許文献２では、「とよのか」特異的マーカーについて２９連鎖群（計109マーカー、全長1451.7cM）「宝交早生」特異的マーカーについて２１連鎖群（計88マーカー、全長1205.7cM）が得られたとあり、うどんこ病発病調査の結果からQTL解析を行ったことが開示されている。ただし、非特許文献２によれば、有望と考えられた連鎖群でLOD値は1.22程度である。 Non-Patent Document 2 discloses that a linkage map was created using hybrid individuals of the strawberry cultivar “Toyooka” × “Takarakosei” and a powdery mildew resistant DNA marker was selected. In Non-Patent Document 2, 29 linkage groups (total 109 markers, total length 1451.7 cM) for the “Toyonoka” specific marker and 21 linkage groups (total 88 markers, total length 1205.7 cM) for the “Takarakosei” specific marker were obtained. It is disclosed that a QTL analysis was conducted from the results of powdery mildew pathogenesis investigation. However, according to Non-Patent Document 2, the LOD value is about 1.22 in a linkage group considered promising.
　非特許文献３には、イチゴうどんこ病への抵抗性が複数の抵抗性遺伝子の集積によること、イチゴうどんこ病罹病性品種「さちのか」と抵抗性を有するF. virginiana（原種）を用いてDNAマーカー137個による全長1360cM、３０連鎖群の連鎖地図を作成したことが開示されている。非特許文献３によれば、イチゴうどんこ病の検定結果と連鎖地図を用いたQTL解析により３箇所にQTLが位置づけられたとある。 In Non-Patent Document 3, resistance to strawberry powdery mildew is due to the accumulation of multiple resistance genes, strawberry powdery mildew susceptible cultivar "Sachinoka" and F. と virginiana (prototype) having resistance are used. It is disclosed that a linkage map of 30 linkage groups with a total length of 1360 cM by 137 DNA markers was prepared. According to Non-Patent Document 3, QTLs are positioned at three locations by the QTL analysis using strawberry powdery mildew test results and a linkage map.
　ところが、上述したようなイチゴうどんこ病抵抗性に関するDNAマーカー技術は、LOD値や寄与率が高いとは言えず、優れたマーカーとは評価できなかった。 However, the above-described DNA marker technology for strawberry powdery mildew resistance cannot be said to be an excellent marker because it cannot be said to have a high LOD value or contribution rate.
　そこで、本発明は、上述した実情に鑑み、多倍数性で複雑なゲノム構造のイチゴ属植物について多数のDNAマーカーを開発し、これら多数のDNAマーカーを用いることでうどんこ病抵抗性を高精度に判定することができるうどんこ病抵抗性関連マーカー及びその利用方法を提供することを目的とする。 Therefore, in view of the above-mentioned circumstances, the present invention has developed a large number of DNA markers for polyploidy and complicated genome structure of Strawberry genus plants, and using these many DNA markers, the powdery mildew resistance is highly accurate. It is an object of the present invention to provide a powdery mildew resistance-related marker that can be determined by the method and a method for using the marker.
　本発明者らは、上記課題を解決するために鋭意検討を重ねた結果、イチゴ属植物における多数のマーカーを準備し、交雑後代系統における形質発現とマーカーとの連鎖解析によって、うどんこ病抵抗性に連鎖するマーカーを見いだし、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors prepared a number of markers in Strawberry genus plants, and developed the resistance to powdery mildew by analyzing the expression of the traits in the hybrid progeny line and the linkage between the markers. As a result, the present inventors have completed the present invention.
　本発明は以下を包含する。
（１）イチゴ属植物の染色体における配列番号１に示す塩基配列及び配列番号１９に示す塩基配列により挟まれる連続する核酸領域からなる、イチゴ属植物うどんこ病抵抗性関連マーカー。
（２）上記核酸領域は、配列番号１～１９からなる群から選ばれるいずれか１の塩基配列又は当該塩基配列の一部を含むことを特徴とする（１）記載のイチゴ属植物うどんこ病抵抗性関連マーカー。
（３）上記核酸領域は、イチゴ属植物の染色体における配列番号１に示す塩基配列と配列番号７に示す塩基配列とにより挟み込まれる領域に位置することを特徴とする（１）記載のイチゴ属植物うどんこ病抵抗性関連マーカー。
（４）少なくとも一方の親がイチゴ属植物である後代植物の染色体及び/又は当該親のイチゴ属植物の染色体を抽出する工程と、上記で得られた染色体における上記（１）乃至（３）いずれか１に記載のイチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を判定する工程とを含む、うどんこ病抵抗性が向上したイチゴ属植物系統の製造方法。
（５）上記判定する工程では、上記イチゴ属植物うどんこ病抵抗性関連マーカーを特異的に増幅するプライマーを用いた核酸増幅反応により当該イチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を判定することを特徴とする（４）記載のイチゴ属植物系統の製造方法。
（６）上記判定する工程では、上記イチゴ属植物うどんこ病抵抗性関連マーカーに対応するプローブを備えるDNAチップを使用することを特徴とする（４）記載のイチゴ属植物系統の製造方法。
（７）上記後代植物は種子又は幼苗であり、当該種子又は幼苗から染色体を抽出することを特徴とする（４）記載のイチゴ属植物系統の製造方法。 The present invention includes the following.
(1) A strawberry genus plant powdery mildew resistance-related marker comprising a continuous nucleic acid region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 19 in the chromosome of a strawberry genus plant.
(2) The strawberry genus plant powdery mildew described in (1), wherein the nucleic acid region comprises any one of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 19 or a part of the base sequence Resistance-related marker.
(3) The nucleic acid region is located in a region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 7 in the chromosome of the strawberry genus plant. Powdery mildew resistance related marker.
(4) a step of extracting a chromosome of a progeny plant in which at least one parent is a strawberry genus plant and / or a chromosome of the parent strawberry genus plant, and any of the above (1) to (3) in the chromosome obtained above A method for producing a strawberry genus plant line having improved powdery mildew resistance, comprising the step of determining the presence / absence of a marker associated with resistance to powdery mildew genus plant according to claim 1.
(5) In the determining step, presence / absence of the strawberry genus plant powdery mildew resistance-related marker by a nucleic acid amplification reaction using a primer that specifically amplifies the strawberry genus plant powdery mildew resistance-related marker. (4) The manufacturing method of the Strawberry genus plant strain | stump | stock characterized by the above-mentioned.
(6) The method for producing a strawberry genus plant line according to (4), wherein in the determining step, a DNA chip comprising a probe corresponding to the strawberry genus plant powdery mildew resistance-related marker is used.
(7) The method according to (4), wherein the progeny plant is a seed or a seedling, and a chromosome is extracted from the seed or the seedling.
　本明細書は本願の優先権の基礎である日本国特許出願2015-054618号及び日本国特許出願2016-042028号の明細書及び/又は図面に記載される内容を包含する。 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2015-054618 and Japanese Patent Application No. 2016-042028 which are the basis of the priority of the present application.
　本発明によれば、イチゴ属植物における形質の中でもうどんこ病抵抗性に連鎖する新規なイチゴ属植物うどんこ病抵抗性関連マーカーを提供することができる。本発明に係るイチゴ属植物うどんこ病抵抗性関連マーカーを利用することによって、イチゴ属植物の交配系統におけるうどんこ病抵抗性を検定することができる。これにより、うどんこ病抵抗性が向上した特性を有するイチゴ属植物系統を非常に低コストに識別することができる。 According to the present invention, it is possible to provide a novel strawberry genus plant powdery mildew resistance-related marker linked to powdery mildew resistance among traits in strawberry plants. By using the strawberry genus plant powdery mildew resistance-related marker according to the present invention, powdery mildew resistance in a hybrid strain of strawberry genus plants can be tested. Thereby, the Strawberry genus plant line which has the characteristic which powdery mildew resistance improved can be identified at very low cost.
イチゴ属植物染色体のマーカーを得る際に使用したDNAマイクロアレイの製造フローを示す模式図である。It is a schematic diagram which shows the manufacture flow of the DNA microarray used when obtaining the marker of the strawberry genus plant chromosome. DNAマイクロアレイを使用したシグナル検出の工程を示す模式図である。It is a schematic diagram which shows the process of the signal detection using a DNA microarray. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代についてイチゴうどんこ病の発症程度を調査した結果を示す特性図である。It is a characteristic figure which shows the result of having investigated the onset degree of the strawberry powdery mildew about the progeny of hybridization of "Miyazaki Natsuharu" and "08 and -f". うどんこ病抵抗性に関するQTL解析の結果（「０８と－ｆ」における第１連鎖群）を示す特性図である。It is a characteristic figure which shows the result (1st linkage group in "08 and -f") of the QTL analysis regarding powdery mildew resistance. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）、「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、うどんこ病発症程度を調査した結果を示す特性図である。"Miyazaki Natsuka Haruka" and "08 and -f" hybrid progeny (Group A), "Miyazaki Natsuka Haruka" and "Ookimi" hybrid progeny (Group B), and "Miyazaki Natsuka Haruka" and "09s Eb It is a characteristic figure which shows the result of having investigated the powdery mildew onset degree about the progeny (E group) of hybridization with "45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）、「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、うどんこ病発症程度を調査した結果を示す特性図である。"Miyazaki Natsuka Haruka" and "08 and -f" hybrid progeny (Group A), "Miyazaki Natsuka Haruka" and "Ookimi" hybrid progeny (Group B), and "Miyazaki Natsuka Haruka" and "09s Eb It is a characteristic figure which shows the result of having investigated the powdery mildew onset degree about the progeny (E group) of hybridization with "45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）における、イチゴうどんこ病抵抗性関連マーカーのアレイシグナル値とＡ集団の表現型とを比較した結果を示す特性図である。A characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）における、イチゴうどんこ病抵抗性関連マーカーのアレイシグナル値とＡ集団の表現型とを比較した結果を示す特性図である。A characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）における、イチゴうどんこ病抵抗性関連マーカーのアレイシグナル値とＡ集団の表現型とを比較した結果を示す特性図である。A characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）における、イチゴうどんこ病抵抗性関連マーカーのアレイシグナル値とＡ集団の表現型とを比較した結果を示す特性図である。A characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）における、イチゴうどんこ病抵抗性関連マーカーのアレイシグナル値とＡ集団の表現型とを比較した結果を示す特性図である。A characteristic diagram showing the results of comparing the array signal value of the strawberry powdery mildew resistance-related marker and the phenotype of the A population in the progeny of the cross between “Mizuki Miyazaki Haruka” and “08 and -f” (A population). is there. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。FIG. 5 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB535110 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and −f”. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。FIG. 5 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB535110 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and −f”. 「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (B group) of hybridization of "Miyazaki Natsuka Haruka" and "Ookimi". 「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (B group) of hybridization of "Miyazaki Natsuka Haruka" and "Ookimi". 「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (E group) of "Miyazaki Natsuka Haruka" and "09s E-b 45e". 「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (E group) of "Miyazaki Natsuka Haruka" and "09s E-b 45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）、「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果をまとめた特性図である。"Miyazaki Natsuka Haruka" and "08 and -f" hybrid progeny (Group A), "Miyazaki Natsuka Haruka" and "Ookimi" hybrid progeny (Group B), and "Miyazaki Natsuka Haruka" and "09s Eb It is the characteristic figure which put together the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (E group) of hybridization with "45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）、「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB535110を特異的に増幅するプライマーを用いたPCRの結果をまとめた特性図である。"Miyazaki Natsuka Haruka" and "08 and -f" hybrid progeny (Group A), "Miyazaki Natsuka Haruka" and "Ookimi" hybrid progeny (Group B), and "Miyazaki Natsuka Haruka" and "09s Eb It is the characteristic figure which put together the result of PCR using the primer which specifically amplifies marker IB535110 about the progeny (E group) of hybridization with "45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。FIG. 6 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB522828 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and −f”. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。FIG. 6 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB522828 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and −f”. 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。FIG. 6 is an electrophoresis photograph showing the results of PCR using a primer that specifically amplifies the marker IB522828 for the progeny (A population) of “Miyazaki Natsuka Haruka” and “08 and −f”. 「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (B group) of a hybrid between "Miyazaki Natsuka Haruka" and "Ookimi". 「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is an electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (B group) of a hybrid between "Miyazaki Natsuka Haruka" and "Ookimi". 「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is the electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (E group) of "Miyazaki Natsuka Haruka" and "09s E-b 45e". 「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果を示す電気泳動写真である。It is the electrophoresis photograph which shows the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (E group) of "Miyazaki Natsuka Haruka" and "09s E-b 45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）、「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果をまとめた特性図である。"Miyazaki Natsuka Haruka" and "08 and -f" hybrid progeny (Group A), "Miyazaki Natsuka Haruka" and "Ookimi" hybrid progeny (Group B), and "Miyazaki Natsuka Haruka" and "09s Eb It is the characteristic figure which put together the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (E group) of hybridization with "45e". 「みやざきなつはるか」と「０８と－ｆ」との交雑後代（Ａ集団）、「みやざきなつはるか」と「おおきみ」との交雑後代（Ｂ集団）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代（Ｅ集団）について、マーカーIB522828を特異的に増幅するプライマーを用いたPCRの結果をまとめた特性図である。"Miyazaki Natsuka Haruka" and "08 and -f" hybrid progeny (Group A), "Miyazaki Natsuka Haruka" and "Ookimi" hybrid progeny (Group B), and "Miyazaki Natsuka Haruka" and "09s Eb It is the characteristic figure which put together the result of PCR using the primer which specifically amplifies marker IB522828 about the progeny (E group) of hybridization with "45e".
　以下、本発明に係るイチゴ属植物うどんこ病抵抗性関連マーカー及びその利用方法、特にイチゴ属植物うどんこ病抵抗性関連マーカーを用いたイチゴ属植物系統の製造方法について説明する。 Hereinafter, the strawberry genus plant powdery mildew resistance-related marker according to the present invention and a method of using the marker, particularly the strawberry genus plant strain-related production method using the strawberry genus plant powdery mildew resistance-related marker will be described.
＜イチゴ属植物うどんこ病抵抗性関連マーカー＞
　本発明に係るイチゴ属植物うどんこ病抵抗性関連マーカーとは、イチゴ属植物の染色体上に存在する特定の領域であり、イチゴ属植物うどんこ病抵抗性という形質を判別できる機能を有する。すなわち、既知のイチゴ属植物を用いて得られた後代系統において、イチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を確認することで、うどんこ病抵抗性を有する系統であるか否かを判断することができる。なお、本発明において、イチゴうどんこ病とは、Ann. Phytopathol. Soc. Jpn. 64:121-l24, l998に記載されるように、Sphaerotheca aphanis（Podosphaera aphanis）が感染することに起因して病班が形成される病気を意味している。 <Strawberry genus plant powdery mildew resistance-related marker>
The strawberry genus plant powdery mildew resistance-related marker according to the present invention is a specific region present on the chromosome of a strawberry genus plant, and has a function of distinguishing the trait of strawberry genus plant powdery mildew resistance. That is, in the progeny line obtained using a known strawberry genus plant, by confirming the presence / absence of the strawberry genus plant powdery mildew resistance-related marker, whether the line has resistance to powdery mildew Can be determined. In the present invention, strawberry powdery mildew is a disease caused by infection of Sphaerotheca aphanis (Podosphaera aphanis) as described in Ann. Phytopathol. Soc. Jpn. 64: 121-l24, l998. It means the disease that the group is formed.
　また、本発明において、イチゴうどんこ病とは、特に、「とよのか」、「女峰」、「麗紅」、「ひみこ」、「宝交早生」、「ダナー」、「幸玉」、「はるのか」及び「福羽」の９品種のうち「とよのか」及び「はるのか」に病原性を示さず他の７品種に病原性を示す菌株（レース0、日本植物病理学会報、第63巻、第3号、第226頁参照）の感染を原因とする病気とすることが好ましい。 Further, in the present invention, strawberry powdery mildew particularly means “Toyonoka”, “Nyoho”, “Rei”, “Himiko”, “Treasure early life”, “Danner”, “Sachitama”, Among the 9 varieties of “Haruno” and “Fukuha”, strains that show no pathogenicity in “Toyonoka” and “Haruno”, but are pathogenic in the other 7 varieties Report 63, No. 3, page 226).
　イチゴ属植物うどんこ病抵抗性関連マーカーとは、うどんこ病抵抗性が高い形質に連鎖するマーカーの意味である。例えば、特定のイチゴ属植物において、イチゴ属植物うどんこ病抵抗性関連マーカーが存在していればうどんこ病抵抗性が高い品種と判断できる。特に、イチゴ属植物うどんこ病抵抗性関連マーカーは、イチゴ属植物のうどんこ病抵抗性といった形質の原因遺伝子（群）に連鎖した領域と考えられる。 Strawberry plant powdery mildew resistance-related marker means a marker linked to a trait that is highly resistant to powdery mildew. For example, in a specific strawberry genus plant, if a strawberry genus plant powdery mildew resistance-related marker exists, it can be determined that the variety has high powdery mildew resistance. In particular, the strawberry genus plant powdery mildew resistance-related marker is considered to be a region linked to a causative gene (group) of traits such as powdery mildew resistance of strawberry plants.
　ここで、イチゴ属植物とは、バラ科イチゴ属(Fragaria L.)に属する植物の全てを含む意味である。すなわち、イチゴ属植物としては、一般的な栽培イチゴであるオランダイチゴ(Fragaria × ananassa)等の交雑種を挙げることができる。また、イチゴ属植物としては、栽培イチゴの祖先種となるF. virginiana並びにF. chiloensis、エゾクサイチゴ（F. vesca）、ノウゴウイチゴ（F. iinumae）、シロバナノヘビイチゴ（F. nipponica）、F. nilgerrensis、F. nubicola、F. bucharica、F. daltoniana、F. orientalis、F. corimbosa、F. moschata及びF. iturupensis等の野生種を挙げることができる。さらに、イチゴ属植物としては、栽培イチゴ（F. × ananassa）における既知の品種・系統も含む意味である。栽培イチゴにおける既知の品種・系統としては、特に限定されず、日本国内にて使用可能なあらゆる品種・系統、日本国外において使用されている品種・系統等を含む意味である。例えば、栽培イチゴの日本国内育成品種としては、特に限定されないが、とよのか、サンチーゴ、純ベリー、女峰、ピーストロ、リンダモール、とちおとめ、アイストロ、栃の峰、章姫、紅ほっぺ、とちひめ、さちのか、けいきわせ、さがほのか、アイベリー、カレンベリー、レッドパール、さつまおとめ、福岡S6（あまおう）、濃姫、ひのみね及び宝交早生等を挙げることができる。 Here, the strawberry genus plant is meant to include all plants belonging to the genus Fragaria L. That is, examples of the Strawberry genus plant include hybrids such as Dutch strawberry (Fragaria × ananassa) which is a general cultivated strawberry. Strawberry genus plants include F. virginiana and F. chiloensis, Ezoxa strawberry (F. iinumae), white snake strawberry (F. nipponica), F. nilgerrensis, which are ancestors of cultivated strawberries. And wild species such as F.colanubicola, F. bucharica, F. daltoniana, F. orientalis, F. corimbosa, F. moschata and F. iturupensis. Furthermore, as a strawberry genus plant, it is the meaning also including the known varieties and strains in cultivated strawberries (F. F x ananassa). Known varieties / lines of cultivated strawberries are not particularly limited, and include all varieties / lines that can be used in Japan, varieties / lines that are used outside of Japan, and the like. For example, cultivated strawberry cultivated varieties in Japan are not particularly limited, but toyoka, Santigo, pure berry, Nyoho, Peace Toro, Linda Mall, Tochiotome, Ice Toro, Tochinomine, Akihime, Beni Hoppe, Tochi Hime, Sachinoka, Keikise, Sagahonoka, Iberry, Karenberry, Red Pearl, Otome Satsuma, Fukuoka S6 (Amaou), Ninohime, Hinone, and Hyocho Precocious.
　イチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を確認する対象の植物としては、上述したイチゴ属植物、上述したイチゴ属植物を利用した後代系統とすることができる。後代系統は、母本及び父本のいずれか一方が上述したイチゴ属植物であればよく、いわゆる同種交配による系統であっても良いし、種間交雑系統であっても良い。また、後代系統は、いわゆる戻し交配によって得られたものでも良い。 As the target plant for confirming the presence / absence of the strawberry genus plant powdery mildew resistance-related marker, the above-mentioned strawberry genus plant and the progeny lineage using the above-mentioned strawberry genus plant can be used. A progeny line | system | group should just be a strawberry genus plant in which any one of a mother book and a father's book mentioned above, the system | strain by what is called a homozygous crossing, and a cross-species hybrid system | strain may be sufficient as it. The progeny line may be obtained by so-called backcrossing.
　特に、栽培イチゴ（F. × ananassa）を対象として、イチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を確認することが好ましい。さらに、栽培イチゴの中でも上述した各種の品種・系統を用いた品種改良において、イチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を確認することが好ましい。すなわち、この場合、作出した新品種についてイチゴうどんこ病抵抗性を判別することができる。よって、新品種としては、イチゴうどんこ病抵抗性を有する品種を少なくとも一方の親として得た品種であることが好ましい。 In particular, it is preferable to confirm the presence / absence of a strawberry genus plant powdery mildew resistance-related marker for cultivated strawberries (F. x ananassa). Furthermore, it is preferable to confirm the presence / absence of a strawberry genus plant powdery mildew resistance-related marker in cultivar improvement using the various varieties / lines described above among cultivated strawberries. That is, in this case, the strawberry powdery mildew resistance can be determined for the produced new variety. Therefore, it is preferable that the new variety is a variety obtained from at least one parent having a strawberry powdery mildew resistance.
　本発明に係るイチゴ属植物うどんこ病抵抗性関連マーカーは、イチゴ品種「みやざきなつはるか」から取得した8,218個のマーカー及びイチゴ系統「０８と－ｆ」から取得した8,039個のマーカーを含む遺伝子連鎖地図と、イチゴうどんこ病抵抗性データとを用いたQTL（Quantitative Trait Loci）解析によって新たに同定されたものである。QTL解析には、遺伝解析ソフトQTL Cartographer（Wang S., C. J. Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC）を使用し、Composite interval mapping（CIM）法を適用している。 The strawberry genus plant powdery mildew resistance-related marker according to the present invention is a gene linkage comprising 8,218 markers obtained from the strawberry cultivar “Miyazaki Natsuka Haruka” and 8,039 markers obtained from the strawberry line “08 and -f”. It was newly identified by QTL (Quantitative Trait Loci) analysis using a map and strawberry powdery mildew resistance data. Genetic analysis software QTL Cartographer (Wang S., C. J. Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC) The Composite interval mapping (CIM) method is applied.
　具体的に、上述したQTL解析により、ロッドスコア（LOD score）が所定の閾値（例えば2.5）以上となる領域を上記遺伝子連鎖地図に見いだした。ピークから1LOD下がる領域は約6.8cM（センチモルガン）であり、「０８と－ｆ」の第１連鎖群に含まれる領域であった。ここで、「モルガン（Ｍ）」は、染色体上の遺伝子間の距離を相対的に示した単位であり、交叉価をパーセントにした値である。イチゴ属植物の染色体において、1cMは、約400kbに相当する。なお、この領域には、ロッドスコアが約7.3のピークが存在しており、当該ピーク位置又はその近傍にイチゴ属植物うどんこ病抵抗性を向上させる形質の原因遺伝子（群）が存在することが示唆される。 Specifically, by the above-described QTL analysis, a region where the rod score (LOD score) is a predetermined threshold (for example, 2.5) or more was found in the gene linkage map. The region 1 LOD down from the peak was about 6.8 cM (centiorgan), which was a region included in the first linkage group of “08 and −f”. Here, “Morgan (M)” is a unit that relatively indicates the distance between genes on a chromosome, and is a value obtained by setting the cross value as a percentage. In the chromosome of a strawberry genus plant, 1 cM corresponds to about 400 kb. In this region, there is a peak having a rod score of about 7.3, and a causative gene (group) of a trait that improves resistance to strawberry plant powdery mildew exists at or near the peak position. It is suggested.
　この6.8cMの領域には、表１に示す１９種類のマーカーがこの順で含まれている。なお、表１において、マーカー名とは、本発明で独自に取得したマーカーに付された名称である。 The 19 kinds of markers shown in Table 1 are included in this order in this 6.8 cM region. In Table 1, the marker name is a name given to the marker uniquely acquired in the present invention.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000005
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000005
　すなわち、本発明に係るイチゴ属植物うどんこ病抵抗性関連マーカーは、イチゴ属植物の染色体における配列番号１に示す塩基配列及び配列番号１９に示す塩基配列により挟まれる連続する核酸領域である。ここで、上記6.8cMの領域に含まれるピークは、配列番号１に示す塩基配列からなるマーカー（IB535110）及び配列番号７に示す塩基配列からなるマーカー（IB713087）により挟み込まれる領域に存在している。 That is, the strawberry genus plant powdery mildew resistance-related marker according to the present invention is a continuous nucleic acid region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 19 in the chromosome of the strawberry plant. Here, the peak contained in the region of 6.8 cM is present in a region sandwiched between a marker (IB535110) consisting of the base sequence shown in SEQ ID NO: 1 and a marker (IB713087) consisting of the base sequence shown in SEQ ID NO: 7. .
　表１に示した6.8cMの領域に含まれる連続した核酸領域を、イチゴ属植物うどんこ病抵抗性関連マーカーとして使用することができる。ここで、核酸領域とは、イチゴ属植物の染色体に存在する他の領域との同一性が９５％以下、好ましくは９０％以下、より好ましくは８０％以下、最も好ましくは７０％以下となるような塩基配列からなる領域を意味する。イチゴ属植物うどんこ病抵抗性関連マーカーとなる核酸領域と他の領域との同一性が上記範囲であれば、定法に従って、当該核酸領域を特異的に検出することができる。ここで、同一性の値は、例えばＢＬＡＳＴを用いてデフォルトパラメータを用いて算出することができる。 The continuous nucleic acid region contained in the 6.8 cM region shown in Table 1 can be used as a marker associated with resistance to powdery mildew plant genus. Here, the nucleic acid region is such that the identity with other regions existing in the chromosome of the strawberry genus plant is 95% or less, preferably 90% or less, more preferably 80% or less, and most preferably 70% or less. This means a region consisting of a simple base sequence. If the identity of the nucleic acid region serving as a marker associated with resistance against powdery mildew plant and other regions is within the above range, the nucleic acid region can be specifically detected according to a standard method. Here, the identity value can be calculated using default parameters using, for example, BLAST.
　また、イチゴ属植物うどんこ病抵抗性関連マーカーとなる核酸領域の塩基長は、少なくとも８塩基長以上、好ましくは１５塩基長以上、より好ましくは２０塩基長以上、最も好ましくは３０塩基長とすることができる。イチゴ属植物うどんこ病抵抗性関連マーカーとなる核酸領域の塩基長が上記範囲であれば、定法に従って、当該核酸領域を特異的に検出することができる。 In addition, the base length of the nucleic acid region serving as a strawberry genus plant powdery mildew resistance-related marker is at least 8 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and most preferably 30 bases in length. be able to. If the base length of the nucleic acid region serving as a strawberry genus plant powdery mildew resistance-related marker is within the above range, the nucleic acid region can be specifically detected according to a conventional method.
　特に、イチゴ属植物うどんこ病抵抗性関連マーカーとしては、上記6.8cMの領域に含まれる１９種類のマーカーのうち、配列番号１に示す塩基配列と配列番号７に示す塩基配列とにより挟み込まれる領域から選ばれることが好ましい。上記ピークが配列番号１に示す塩基配列と配列番号７に示す塩基配列とにより挟み込まれる領域に存在するためである。 In particular, the strawberry genus plant powdery mildew resistance-related marker is a region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 7 among 19 types of markers contained in the 6.8 cM region. Is preferably selected from. This is because the peak exists in a region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 7.
　また、イチゴ属植物うどんこ病抵抗性関連マーカーとしては、上記表１に示した１９種類のマーカーから選ばれる１種類のマーカーを含む核酸領域とすることもできる。例えば、イチゴ属植物うどんこ病抵抗性関連マーカーとしては、ピークの位置に最も近い配列番号１に示す塩基配列からなるマーカー（IB535110）を含む核酸領域を使用することが好ましい。このとき、マーカーを含む核酸領域の塩基配列は、当該マーカーの塩基配列に基づいて設計したプライマーを用いたインバースPCR等の隣接配列取得法によって特定することができる。 Alternatively, the strawberry genus plant powdery mildew resistance-related marker may be a nucleic acid region containing one type of marker selected from the 19 types of markers shown in Table 1 above. For example, as a strawberry genus plant powdery mildew resistance-related marker, it is preferable to use a nucleic acid region containing a marker (IB535110) consisting of the base sequence shown in SEQ ID NO: 1 closest to the peak position. At this time, the base sequence of the nucleic acid region containing the marker can be specified by an adjacent sequence acquisition method such as inverse PCR using a primer designed based on the base sequence of the marker.
　さらに、イチゴ属植物の染色体における配列番号１に示す塩基配列及び配列番号１９に示す塩基配列により挟まれる核酸領域から、複数の領域をイチゴ属植物うどんこ病抵抗性関連マーカーとすることもできる。 Furthermore, a plurality of regions can be used as a marker associated with resistance to powdery mildew in the strawberry genus plant from the nucleic acid region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 19 in the chromosome of the strawberry genus plant.
　さらにまた、イチゴ属植物うどんこ病抵抗性関連マーカーとしては、上記１９種類のマーカーそのものを使用することができる。すなわち、配列番号１～１９の塩基配列からなる１９個の領域から選ばれる１又は複数の領域をイチゴ属植物うどんこ病抵抗性関連マーカーとすることができる。例えば、イチゴ属植物うどんこ病抵抗性関連マーカーとしては、ピークの位置に最も近い配列番号１に示す塩基配列からなるマーカー（IB535110）を使用することが好ましい。或いは、例えば、配列番号２の塩基配列からなるマーカー（IB522828）と配列番号3の塩基配列からなるマーカー（IB559302）とで挟み込まれる領域をイチゴ属植物うどんこ病抵抗性関連マーカーとすることもできる。 Furthermore, the above 19 kinds of markers themselves can be used as the strawberry genus plant powdery mildew resistance-related marker. That is, one or a plurality of regions selected from the 19 regions consisting of the nucleotide sequences of SEQ ID NOS: 1 to 19 can be used as a strawberry genus plant powdery mildew resistance-related marker. For example, as a strawberry genus plant powdery mildew resistance-related marker, it is preferable to use a marker (IB535110) comprising the base sequence shown in SEQ ID NO: 1 closest to the peak position. Alternatively, for example, a region sandwiched between a marker consisting of the base sequence of SEQ ID NO: 2 (IB522828) and a marker consisting of the base sequence of SEQ ID NO: 3 (IB559302) can be used as a strawberry plant powdery mildew resistance-related marker. .
＜イチゴ属植物におけるマーカーの同定＞
　本発明では、上述したように、イチゴ品種「みやざきなつはるか」から取得した8,218個のマーカー及びイチゴ系統「０８と－ｆ」から取得した8,039個のマーカーからイチゴ属植物うどんこ病抵抗性関連マーカーを特定した。ここでは、これら8,218個と8,039個のマーカーについて説明する。このマーカーを同定する際には、特開2011-120558号公報や国際公開公報2011/074510に開示された方法を適用したDNAマイクロアレイを使用することができる。 <Identification of markers in strawberry plants>
In the present invention, as described above, the strawberry genus plant powdery mildew resistance-related marker is obtained from 8,218 markers obtained from the strawberry cultivar “Miyazaki Natsuka Haruka” and 8,039 markers obtained from the strawberry line “08 and -f”. Identified. Here, these 8,218 and 8,039 markers will be described. When this marker is identified, a DNA microarray to which the methods disclosed in JP 2011-120558 A and International Publication 2011/074510 are applied can be used.
　具体的に、当該DNAマイクロアレイに使用するプローブの設計方法は、図１に示すように、先ず、「みやざきなつはるか」或いは「０８と－ｆ」からゲノムDNAを抽出する（工程1a）。次に、抽出したゲノムDNAを１又は複数の制限酵素により消化する（工程1b）。なお、図１に示した例では、制限酵素Ａ及び制限酵素Ｂの２種類の制限酵素をこの順で用いてゲノムDNAを消化している。ここで、制限酵素としては、特に限定されないが、例えば、PstI、EcoRI、HindIII、BstNI、HpaII、HaeIII等を使用することができる。特に制限酵素としては、ゲノムDNAを完全に消化した際に20～10000塩基長のゲノムDNA断片となるよう、認識配列の出現頻度等を考慮して適宜選択することができる。また、複数の制限酵素を使用する場合、全ての制限酵素を使用した後のゲノムDNA断片が200～6000塩基長となっていることが好ましい。さらに、複数の制限酵素を使用する場合、処理に供する制限酵素の順序は特に限定されず、また、処理条件（溶液組成や温度等）が共通する場合には複数の制限酵素を同一の反応系で使用しても良い。すなわち、図１に示した例においては、制限酵素Ａ及び制限酵素Ｂをこの順で使用してゲノムDNAを消化しているが、制限酵素Ａ及び制限酵素Ｂを同じ反応系で同時に使用してゲノムDNAを消化しても良いし、制限酵素Ｂ及び制限酵素Ａをこの順で使用してゲノムDNAを消化してもよい。さらに、使用する制限酵素の数は３以上であってもよい。 Specifically, as shown in FIG. 1, the design method of the probe used for the DNA microarray first extracts genomic DNA from “Miyazaki Natsuki Haruka” or “08 and -f” (step 1a). Next, the extracted genomic DNA is digested with one or more restriction enzymes (step 1b). In the example shown in FIG. 1, genomic DNA is digested using two types of restriction enzymes, restriction enzyme A and restriction enzyme B, in this order. Here, the restriction enzyme is not particularly limited, and for example, PstI, EcoRI, HindIII, BstNI, HpaII, HaeIII and the like can be used. In particular, the restriction enzyme can be appropriately selected in consideration of the appearance frequency of the recognition sequence so that a genomic DNA fragment having a length of 20 to 10,000 bases is obtained when the genomic DNA is completely digested. When a plurality of restriction enzymes are used, it is preferable that the genomic DNA fragment after using all restriction enzymes has a length of 200 to 6000 bases. Furthermore, when using a plurality of restriction enzymes, the order of restriction enzymes to be used for the treatment is not particularly limited, and when the processing conditions (solution composition, temperature, etc.) are common, a plurality of restriction enzymes are used in the same reaction system. May be used in That is, in the example shown in FIG. 1, restriction enzyme A and restriction enzyme B are used in this order to digest genomic DNA, but restriction enzyme A and restriction enzyme B are simultaneously used in the same reaction system. Genomic DNA may be digested, or genomic DNA may be digested using restriction enzyme B and restriction enzyme A in this order. Further, the number of restriction enzymes used may be 3 or more.
　次に、制限酵素処理後のゲノムDNA断片に対してアダプターを結合する（工程1c）。ここで、アダプターとは、上述した制限酵素処理によって得られたゲノムDNA断片の両端に結合できるものであれば特に限定されない。アダプターとしては、例えば、制限酵素処理によってゲノムDNAの両末端に形成される突出末端（粘着末端）に対して相補的な一本鎖を有し、詳細を後述する増幅処理の際に使用するプライマーがハイブリダイズしうるプライマー結合配列を有するものを使用することができる。また、アダプターとしては、上記突出末端（粘着末端）に対して相補的な一本鎖を有し、クローニングする際のベクターに組み入れるための制限酵素認識部位を有するものを使用することもできる。 Next, an adapter is bound to the genomic DNA fragment after the restriction enzyme treatment (step 1c). Here, the adapter is not particularly limited as long as it can bind to both ends of the genomic DNA fragment obtained by the restriction enzyme treatment described above. As an adapter, for example, a primer that has a single strand complementary to the protruding ends (sticky ends) formed at both ends of genomic DNA by restriction enzyme treatment, and is used in the amplification process described in detail later Having a primer binding sequence capable of hybridizing can be used. As the adapter, one having a single strand complementary to the protruding end (adhesive end) and having a restriction enzyme recognition site for incorporation into a vector for cloning can also be used.
　また、複数の制限酵素を使用してゲノムDNAを消化した場合には、各制限酵素に対応する複数のアダプターを準備して使用することができる。すなわち、複数の制限酵素でゲノムDNAを消化した場合に生ずる複数種類の突出末端のそれぞれに対して、相補的な一本鎖を有する複数のアダプターを使用することができる。このとき、複数の制限酵素に対応する複数のアダプターは、共通するプライマーがハイブリダイズできるように共通するプライマー結合配列を有しているものであっても良いし、それぞれ異なるプライマーがハイブリダイズできるように異なるプライマー結合配列を有するものであっても良い。 In addition, when genomic DNA is digested using a plurality of restriction enzymes, a plurality of adapters corresponding to each restriction enzyme can be prepared and used. That is, a plurality of adapters having a single strand complementary to each of a plurality of types of protruding ends generated when genomic DNA is digested with a plurality of restriction enzymes can be used. At this time, the plurality of adapters corresponding to the plurality of restriction enzymes may have a common primer binding sequence so that a common primer can hybridize, or different primers can hybridize with each other. May have different primer binding sequences.
　さらに、複数の制限酵素を使用してゲノムDNAを消化した場合、アダプターとしては、使用した複数の制限酵素のなかから選ばれる１つの制限酵素若しくは、使用した制限酵素のうち一部の制限酵素に対応するアダプターを準備して使用することもできる。 Furthermore, when digesting genomic DNA using a plurality of restriction enzymes, the adapter may be one restriction enzyme selected from the plurality of restriction enzymes used or a part of the restriction enzymes used. A corresponding adapter can also be prepared and used.
　次に、両末端にアダプターが付加されたゲノムDNA断片を増幅する（工程1d）。プライマー結合配列を有するアダプターを使用した場合には、当該プライマー結合配列にハイブリダイズできるプライマーを使用することで上記ゲノムDNA断片を増幅することができる。或いは、アダプターを付加したゲノムDNA断片を、アダプター配列を利用してベクターにクローニングし、当該ベクターにおける所定の領域にハイブリダイズできるプライマーを用いてゲノムDNA断片を増幅することができる。なお、プライマーを用いたゲノムDNA断片の増幅反応としては、一例としてPCRを使用することができる。 Next, a genomic DNA fragment with adapters added to both ends is amplified (step 1d). When an adapter having a primer binding sequence is used, the genomic DNA fragment can be amplified by using a primer that can hybridize to the primer binding sequence. Alternatively, a genomic DNA fragment to which an adapter has been added can be cloned into a vector using the adapter sequence, and the genomic DNA fragment can be amplified using a primer that can hybridize to a predetermined region in the vector. In addition, PCR can be used as an example of the amplification reaction of a genomic DNA fragment using a primer.
　また、複数の制限酵素を使用してゲノムDNAを消化するとともに、各制限酵素に対応する複数のアダプターをゲノムDNA断片に連結した場合、複数の制限酵素を用いた処理によって得られたゲノムDNA断片の全てにアダプターが連結されることとなる。この場合、アダプターに含まれるプライマー結合配列を用いて核酸増幅反応を行うことで、得られた全てのゲノムDNA断片を増幅することができる。 In addition, when digesting genomic DNA using multiple restriction enzymes and ligating multiple adapters corresponding to each restriction enzyme to genomic DNA fragments, genomic DNA fragments obtained by treatment with multiple restriction enzymes An adapter will be connected to all of these. In this case, all the genomic DNA fragments obtained can be amplified by performing a nucleic acid amplification reaction using the primer binding sequence contained in the adapter.
　或いは、複数の制限酵素を使用してゲノムDNAを消化するとともに、使用した複数の制限酵素のなかから選ばれる１つの制限酵素若しくは、使用した制限酵素のうち一部の制限酵素に対応するアダプターをゲノムDNA断片に連結した場合、得られたゲノムDNA断片のうち、選ばれた制限酵素の認識配列を両末端に有するゲノムDNA断片のみを増幅することができる。 Alternatively, genomic DNA is digested using a plurality of restriction enzymes, and one restriction enzyme selected from the plurality of restriction enzymes used or an adapter corresponding to a part of the restriction enzymes used is used. When ligated to a genomic DNA fragment, only the genomic DNA fragment having a recognition sequence of a selected restriction enzyme at both ends can be amplified from the obtained genomic DNA fragment.
　次に、増幅されたゲノムDNA断片の塩基配列を決定し（工程1e）、当該ゲノムDNA断片より短い塩基長を有し、ゲノムDNA断片内の少なくとも一部をカバーする１又は複数の領域を特定し、特定した１又は複数の領域を、栽培いちごにおけるプローブとして設計する（工程1f）。ゲノムDNA断片の塩基配列を決定する方法は、特に限定されず、サンガー法等を適用したDNAシークエンサーを利用した従来公知の方法を使用することができる。ここで、設計する領域としては、上述したように、例えば20～100塩基長、好ましくは30～90塩基長、より好ましくは50～75塩基長とする。 Next, the base sequence of the amplified genomic DNA fragment is determined (step 1e), and one or more regions having a base length shorter than the genomic DNA fragment and covering at least a part of the genomic DNA fragment are identified. Then, the specified region or regions are designed as probes in the cultivated strawberry (step 1f). The method for determining the base sequence of the genomic DNA fragment is not particularly limited, and a conventionally known method using a DNA sequencer to which the Sanger method or the like is applied can be used. Here, the region to be designed is, for example, 20 to 100 bases long, preferably 30 to 90 bases long, more preferably 50 to 75 bases long as described above.
　以上のように、栽培いちごから抽出したゲノムDNAを使用して多数のプローブを設計し、設計したプローブの塩基配列に基づいて、担体上にて目的の塩基配列を有するオリゴヌクレオチドを合成することでDNAマイクロアレイを作製することができる。このように作製したDNAマイクロアレイを使用することで、上述した配列番号１～１９に示した１９種類のイチゴ属植物うどんこ病抵抗性関連マーカーを含む8,218個と8,039個のマーカーを同定することができる。 As described above, a large number of probes are designed using genomic DNA extracted from cultivated strawberries, and an oligonucleotide having the desired base sequence is synthesized on a carrier based on the base sequence of the designed probe. DNA microarrays can be made. By using the thus prepared DNA microarray, it is possible to identify 8,218 and 8,039 markers including 19 kinds of strawberry plant powdery mildew resistance-related markers shown in SEQ ID NOS: 1 to 19 above. it can.
　より具体的に、本発明者らは、イチゴ品種「みやざきなつはるか」から取得した8,218個のマーカー、イチゴ系統「０８と－ｆ」及びこれらの交配後代系統（147系統）について、上述したDNAマイクロアレイを用いてシグナルデータを取得した。そして、得られたシグナルデータから遺伝子型データを取得し、この遺伝子型データを元にして、遺伝地図作成ソフトウェアAntMap（Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm. Breed Sci 56: 371-378）を使用し、遺伝距離計算式Kosambiにより染色体におけるマーカーの位置情報を算出した。さらに、取得したマーカーの位置情報をもとに、Mapmaker/EXP ver.3.0（A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993）により遺伝地図データシートを作成した。その結果、上述した配列番号１～１９に示した１９種類のイチゴ属植物うどんこ病抵抗性関連マーカーを含む8,218個と8,039個のマーカーを同定している。 More specifically, the present inventors have described the DNA microarray described above for 8,218 markers obtained from the strawberry cultivar “Miyazaki Natsuka Haruka”, strawberry lines “08 and -f” and their progeny lines (147 lines). Signal data was acquired using. And genotype data is obtained from the obtained signal data, and based on this genotype data, genetic map creation software AntMap (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm (Breed Sci 56: 371-378) was used to calculate the position information of the marker on the chromosome using the genetic distance calculation formula Kosambi. Furthermore, based on the acquired marker position information, a genetic map data sheet was prepared by Mapmaker / EXP ver.3.0 (A Whitehead Institute Institute for Biomedical Research Research Report, Third Edition, January 1993). As a result, 8,218 and 8,039 markers including 19 kinds of strawberry genus plant powdery mildew resistance-related markers shown in SEQ ID NOs: 1 to 19 were identified.
＜イチゴ属植物うどんこ病抵抗性関連マーカーの利用＞
　イチゴ属植物うどんこ病抵抗性関連マーカーを利用することで、うどんこ病抵抗性が未知であるイチゴ属植物（例えば、後代系統）についてうどんこ病抵抗性を示す系統であるか判断することができる。ここで、イチゴ属植物うどんこ病抵抗性関連マーカーを利用するとは、当該マーカーを含む核酸断片を特異的に増幅する方法を利用する形態、当該マーカーに対応するプローブを有するDNAマイクロアレイを利用する形態を含む意味である。 <Utilization of markers related to resistance to powdery mildew plant genus>
By using a strawberry plant powdery mildew resistance-related marker, it is possible to determine whether a strawberry genus plant (for example, a progeny strain) whose powdery mildew resistance is unknown is a strain exhibiting powdery mildew resistance. it can. Here, using a strawberry genus plant powdery mildew resistance-related marker means a form using a method of specifically amplifying a nucleic acid fragment containing the marker, a form using a DNA microarray having a probe corresponding to the marker It means to include.
　イチゴ属植物うどんこ病抵抗性関連マーカーを含む核酸断片を特異的に増幅する方法とは、いわゆる核酸増幅法を利用することを意味する。核酸増幅法としては、目的とする核酸断片を特異的に増幅するように設計したプライマーを使用する方法、プライマーを使用することなく目的とする核酸断片を特異的に増幅する方法が挙げられる。 The method of specifically amplifying a nucleic acid fragment containing a strawberry genus plant powdery mildew resistance-related marker means using a so-called nucleic acid amplification method. Examples of the nucleic acid amplification method include a method using a primer designed to specifically amplify a target nucleic acid fragment, and a method of specifically amplifying a target nucleic acid fragment without using a primer.
　目的とする核酸断片を特異的に増幅するプライマーとは、核酸増幅法によって、上述のように定義されたイチゴ属植物うどんこ病抵抗性関連マーカーを含む核酸断片を増幅できるオリゴヌクレオチドを意味する。プライマーを使用する核酸増幅法としては、特に限定されず、PCR(Polymerase Chain Reaction)法に代表される核酸断片を増幅させるものであればどのようなものでもかまわない。例えば、PCR法以外に、RCA(Rolling Circle Amplification)法、CPT(Cycling Probe Technology)法、ICAN(Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids)法、LAMP(Loop-Mediated Isothermal Amplificaton of DNA)法、SDA(Strand Displacement Amplification)法、NASBA(Nucleic acid Sequence-based Amplification method)法、及びTMA(Transcription mediated amplification method)法等の公知の方法が例示できるが、これらに限定されるものではない。 A primer that specifically amplifies a target nucleic acid fragment means an oligonucleotide that can amplify a nucleic acid fragment containing a strawberry genus plant powdery mildew resistance-related marker as defined above by a nucleic acid amplification method. The nucleic acid amplification method using a primer is not particularly limited, and any method may be used as long as it amplifies a nucleic acid fragment typified by a PCR (Polymerase Chain Reaction) method. For example, in addition to PCR, RCA (RollingRCircle Amplification) method, CPT (Cycling Probe Technology) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) method, LAMP (Loop-Mediated Isothermal Amplificaton of) Known methods such as SDA (Strand Displacement Amplification) method, NASBA (Nucleic acid Sequence-based Amplification method) method, and TMA (Transcription mediated amplification method) method can be exemplified, but are not limited thereto.
　これら核酸増幅反応のうち例えばPCR法を利用する場合、イチゴ属植物の染色体におけるイチゴ属植物うどんこ病抵抗性関連マーカーを挟み込むように一対のプライマーを設計する。また、LAMP法を利用する場合、イチゴ属植物の染色体におけるイチゴ属植物うどんこ病抵抗性関連マーカーを挟み込むように４種類のプライマーを設計する。 Of these nucleic acid amplification reactions, for example, when using the PCR method, a pair of primers is designed so as to sandwich a strawberry plant powdery mildew resistance-related marker in the chromosome of the strawberry plant. In addition, when the LAMP method is used, four kinds of primers are designed so as to sandwich a strawberry genus plant powdery mildew resistance-related marker in the chromosome of the strawberry genus plant.
　プライマーを使用しない核酸増幅法としては、特に限定されないが、LCR(Ligase Chain Reaction)法を挙げることができる。ただし、LCR法においても、イチゴ属植物うどんこ病抵抗性関連マーカーを含む核酸断片にハイブリダイズする複数のオリゴヌクレオチドを設計する。 The nucleic acid amplification method without using a primer is not particularly limited, and examples thereof include an LCR (LigaseigChain Reaction) method. However, in the LCR method as well, a plurality of oligonucleotides that hybridize to a nucleic acid fragment containing a strawberry genus plant powdery mildew resistance-related marker are designed.
　以上のように、核酸増幅法によれば、検査対象のイチゴ属植物に上記イチゴ属植物うどんこ病抵抗性関連マーカーが存在している場合、当該マーカーを含む核酸断片を増幅産物として得ることができる。言い換えると、検査対象のイチゴ属植物から抽出した染色体を鋳型とした核酸増幅法によって所期の核酸断片が増幅された場合には、当該検査対象のイチゴ属植物がうどんこ病抵抗性を有すると判断することができる。 As described above, according to the nucleic acid amplification method, when the strawberry genus plant powdery mildew resistance-related marker is present in the strawberry genus plant to be examined, a nucleic acid fragment containing the marker can be obtained as an amplification product. it can. In other words, if the desired nucleic acid fragment is amplified by the nucleic acid amplification method using the chromosome extracted from the strawberry plant to be tested as a template, the strawberry genus plant to be tested has powdery mildew resistance. Judgment can be made.
　増幅された核酸断片を検出するには、特に限定されないが、増幅反応後の溶液をアガロース電気泳動にかけ、Ethidium BromideやSYBR Greenなどの蛍光性インターカレーターを結合させ特異的な蛍光を観察する方法、核酸増幅反応の溶液に蛍光性インターカレーターを添加し、増幅反応後の蛍光検出を行う方法、蛍光標識されたプライマーを用いて核酸増幅反応を行い、増幅反応後の蛍光検出を行う方法等を挙げることができる。 To detect the amplified nucleic acid fragment, although not particularly limited, a method for observing specific fluorescence by subjecting the solution after amplification reaction to agarose electrophoresis and binding a fluorescent intercalator such as Ethidium Bromide or SYBR Green, Examples include a method of adding a fluorescent intercalator to a nucleic acid amplification reaction solution to detect fluorescence after the amplification reaction, a method of performing nucleic acid amplification reaction using a fluorescently labeled primer, and detecting fluorescence after the amplification reaction be able to.
　なお、核酸増幅法を利用してイチゴ属植物うどんこ病抵抗性関連マーカーを検出する場合、当該マーカーを含む増幅断片の塩基長は、核酸増幅方法の原理等によっても異なるが、例えば30～10000塩基長とすることができ、50～5000塩基長とすることが好ましく、70～2000塩基長とすることがより好ましい。 When detecting a strawberry genus plant powdery mildew resistance-related marker using a nucleic acid amplification method, the base length of the amplified fragment containing the marker varies depending on the principle of the nucleic acid amplification method, etc., for example, 30 to 10,000. The length can be a base length, preferably 50 to 5000 bases, more preferably 70 to 2000 bases.
　また、イチゴ属植物におけるうどんこ病抵抗性を判定する際、複数のイチゴ属植物うどんこ病抵抗性関連マーカーを検出しても良い。すなわち、イチゴ属植物の染色体における配列番号１に示す塩基配列及び配列番号１９に示す塩基配列により挟まれる核酸領域から選ばれる複数の領域をイチゴ属植物うどんこ病抵抗性関連マーカーとし、これら複数のイチゴ属植物うどんこ病抵抗性関連マーカーを検出しても良い。例えば、配列番号１～１９の塩基配列からなる１９個の領域から選ばれる複数の領域をイチゴ属植物うどんこ病抵抗性関連マーカーとし、これら複数の領域を検出しても良い。 Moreover, when determining the powdery mildew resistance in a strawberry genus plant, a plurality of strawberry genus plant powdery mildew resistance-related markers may be detected. That is, a plurality of regions selected from the nucleic acid region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 19 in the chromosome of the strawberry genus plant is used as a strawberry genus plant powdery mildew resistance-related marker, Strawberry plant powdery mildew resistance-related markers may be detected. For example, a plurality of regions selected from 19 regions consisting of the base sequences of SEQ ID NOs: 1 to 19 may be used as a strawberry genus plant powdery mildew resistance-related marker, and the plurality of regions may be detected.
　一例としては、配列番号１の塩基配列からなる領域（IB535110）と配列番号２の塩基配列からなる領域（IB522828）とをそれぞれイチゴ属植物うどんこ病抵抗性関連マーカーとし、これら各領域を核酸増幅法により増幅してイチゴ属植物うどんこ病抵抗性関連マーカーの存否を確認することができる。或いは、一例として配列番号２の塩基配列からなる領域（IB522828）と配列番号３の塩基配列からなる領域（IB559302）とで挟み込まれる領域をイチゴ属植物うどんこ病抵抗性関連マーカーとし、この領域を核酸増幅法により増幅してイチゴ属植物うどんこ病抵抗性関連マーカーの存否を確認することができる。 As an example, the region consisting of the nucleotide sequence of SEQ ID NO: 1 (IB535110) and the region consisting of the nucleotide sequence of SEQ ID NO: 2 (IB522828) are used as markers associated with powdery mildew plant resistance, respectively, and each of these regions is amplified by nucleic acid. It can be amplified by the method to confirm the presence or absence of a strawberry genus plant powdery mildew resistance-related marker. Alternatively, as an example, a region sandwiched between a region consisting of the base sequence of SEQ ID NO: 2 (IB522828) and a region consisting of the base sequence of SEQ ID NO: 3 (IB559302) is used as a strawberry genus plant powdery mildew resistance related marker, It can be amplified by a nucleic acid amplification method to confirm the presence or absence of a strawberry genus plant powdery mildew resistance-related marker.
　一方、イチゴ属植物うどんこ病抵抗性関連マーカーに対応するプローブを有するDNAマイクロアレイを利用する形態において、当該プローブとは、上述のように定義されたイチゴ属植物うどんこ病抵抗性関連マーカーに対して、ストリンジェントな条件下で特異的にハイブリダイズできるオリゴヌクレオチドを意味する。このようなオリゴヌクレオチドは、例えば、上述のように定義されたイチゴ属植物うどんこ病抵抗性関連マーカーの塩基配列又はその相補鎖の少なくとも連続する１０塩基、１５塩基、２０塩基、２５塩基、３０塩基、３５塩基、４０塩基、４５塩基、５０塩基又はそれ以上の塩基長の部分領域若しくは全領域として設計することができる。なお、このプローブを有するDNAマイクロアレイとしては、ガラスやシリコーン等の平面基板を担体とするマイクロアレイや、マイクロビーズを担体とするビーズアレイ、或いは中空繊維の内壁にプローブを固定する３次元マイクロアレイ等の如何なるタイプのマイクロアレイであってもよい。 On the other hand, in a form using a DNA microarray having a probe corresponding to a strawberry genus plant powdery mildew resistance-related marker, the probe is a strawberry genus plant powdery mildew resistance-related marker defined as described above. An oligonucleotide that can specifically hybridize under stringent conditions. Such oligonucleotides include, for example, at least 10 bases, 15 bases, 20 bases, 25 bases, 30 bases of the base sequence of the strawberry genus plant powdery mildew resistance-related marker defined above or the complementary strand thereof. It can be designed as a partial region or a whole region having a base length of 35, 40, 45, 50 or more bases. The DNA microarray having the probe may be any microarray using a flat substrate such as glass or silicone as a carrier, a bead array using a microbead as a carrier, or a three-dimensional microarray in which a probe is fixed to the inner wall of a hollow fiber. It may be a type of microarray.
　以上のように作製されたDNAマイクロアレイを使用することで、後代系統等に代表されるうどんこ病抵抗性の表現型が未知のイチゴ属植物について、うどんこ病抵抗性に優れるという表現型を示す系統であるか判断することができる。なお、上述したDNAマイクロアレイを使用する方法以外であっても、従来公知の手法を用いて上述したイチゴ属植物うどんこ病抵抗性関連マーカーを検出して、供試イチゴ属植物についてうどんこ病抵抗性に優れるという形質を有する系統であるか判断してもよい。DNAマイクロアレイを使用する方法以外の方法としては、例えば、上述したプローブを用いた、いわゆるFISH（fluorescence in situ hybridization）法を適用することができる。 By using the DNA microarray prepared as described above, strawberry genus plants with unknown powdery mildew resistance phenotypes, such as progeny lines, exhibit a phenotype that is excellent in powdery mildew resistance. It can be judged whether it is a system. In addition to the method using the above-described DNA microarray, the above-mentioned strawberry genus plant powdery mildew resistance-related marker is detected using a conventionally known method, and the powdery mildew resistance of the test strawberry genus plant is detected. You may judge whether it is a strain | stump | stock which has the character of being excellent in property. As a method other than the method using the DNA microarray, for example, a so-called FISH (fluorescence in situ hybridization) method using the above-described probe can be applied.
　DNAマイクロアレイを使用する方法をより詳細に説明する。図２に示すように、先ず供試イチゴ属植物からゲノムDNAを抽出する。この供試イチゴ属植物とは、後代系統等のうどんこ病抵抗性の表現型が未知のイチゴ属植物及び/又は後代系統を作製する際に使用した親のイチゴ属植物のことであり、うどんこ病抵抗性に優れるといった形質を有するか判定する対象となるイチゴ属植物である。 The method of using the DNA microarray will be described in more detail. As shown in FIG. 2, first, genomic DNA is extracted from the strawberry genus plant. This strawberry genus plant is a strawberry genus plant with unknown powdery mildew resistance phenotype and / or a parent strawberry genus plant used when producing a progeny strain, It is a strawberry genus plant for which it is judged whether it has a trait such as excellent disease resistance.
　次に、抽出したゲノムDNAを、上記＜イチゴ属植物におけるマーカーの同定＞の欄で説明したDNAマイクロアレイを作製する際に使用した制限酵素で消化して複数のゲノムDNA断片を調整する。次に、得られたゲノムDNA断片と、DNAマイクロアレイを作製する際に使用したアダプターとを連結する。次に、両末端にアダプターが付加されたゲノムDNA断片を、DNAマイクロアレイを作製する際に使用したプライマーを用いて増幅する。これにより、DNAマイクロアレイを作製する際の工程1dで増幅したゲノムDNA断片に対応する、供試イチゴ属植物由来のゲノムDNA断片を増幅することができる。 Next, the extracted genomic DNA is digested with the restriction enzymes used in preparing the DNA microarray described in the above section <Identification of Markers in Strawberry Plant> to prepare a plurality of genomic DNA fragments. Next, the obtained genomic DNA fragment is ligated to the adapter used in preparing the DNA microarray. Next, the genomic DNA fragment with adapters added to both ends is amplified using the primers used in preparing the DNA microarray. Thereby, the genomic DNA fragment derived from the strawberry genus plant corresponding to the genomic DNA fragment amplified in step 1d when preparing the DNA microarray can be amplified.
　この工程においては、アダプターが付加されたゲノムDNA断片のうち、所定のゲノムDNA断片を選択的に増幅してもよい。例えば、複数の制限酵素に対応する複数のアダプターを使用した場合には、特定のアダプターが付加されたゲノムDNA断片を選択的に増幅することができる。また、複数の制限酵素でゲノムDNAを消化した場合、得られたゲノムDNA断片のうち、所定の制限酵素に対応する突出末端を有するゲノムDNA断片のみにアダプターを付加することで、アダプターが付加されたゲノムDNA断片を選択的に増幅することができる。このように、所定のゲノムDNA断片を選択的に増幅することで濃縮させることができる。 In this step, a predetermined genomic DNA fragment may be selectively amplified among the genomic DNA fragments to which the adapter is added. For example, when a plurality of adapters corresponding to a plurality of restriction enzymes are used, a genomic DNA fragment to which a specific adapter is added can be selectively amplified. In addition, when genomic DNA is digested with multiple restriction enzymes, the adapter is added by adding the adapter only to the genomic DNA fragment having a protruding end corresponding to the predetermined restriction enzyme. Genomic DNA fragments can be selectively amplified. Thus, it can be concentrated by selectively amplifying a predetermined genomic DNA fragment.
　次に、増幅したゲノムDNA断片に標識を付加する。標識としては、従来公知の如何なる物質を使用しても良い。標識としては、例えば蛍光分子、色素分子、放射性分子等を使用することができる。なお、本工程は、ゲノムDNA断片を増幅する工程において標識を有するヌクレオチドを用いることで省略することができる。上記工程において標識を有するヌクレオチドを用いてゲノムDNA断片を増幅することで、増幅されたDNA断片が標識化されるためである。 Next, a label is added to the amplified genomic DNA fragment. Any conventionally known substance may be used as the label. As the label, for example, fluorescent molecules, dye molecules, radioactive molecules and the like can be used. This step can be omitted by using a nucleotide having a label in the step of amplifying the genomic DNA fragment. This is because the amplified DNA fragment is labeled by amplifying the genomic DNA fragment using a nucleotide having a label in the above step.
　次に、標識を有するゲノムDNA断片を所定の条件下でDNAマイクロアレイに接触させ、DNAマイクロアレイに固定されたプローブと標識を有するゲノムDNA断片とをハイブリダイズさせる。このとき、ハイブリダイズさせる際には高いストリンジェンシー条件とすることが好ましい。このような高いストリンジェンシー条件とすることによって、供試イチゴ属植物にイチゴ属植物うどんこ病抵抗性関連マーカーが存在しているか否かを、より高精度に判定することができる。なお、ストリンジェンシー条件は、反応温度及び塩濃度で調節することができる。すなわち、より高温とすることでより高いストリンジェンシー条件となり、またより低い塩濃度でより高いストリンジェンシー条件となる。例えば、50～75塩基長のプローブを使用する場合、ハイブリダイゼーション条件としては、40～44℃、0.2%SDS、6×SSCの条件とすることでより高いストリンジェンシー条件とすることができる。 Next, the genomic DNA fragment having the label is brought into contact with the DNA microarray under predetermined conditions, and the probe immobilized on the DNA microarray and the genomic DNA fragment having the label are hybridized. At this time, it is preferable to use high stringency conditions for hybridization. By setting it as such a high stringency condition, it can be determined more accurately whether the strawberry genus plant powdery mildew resistance related marker exists in a test strawberry genus plant. Stringency conditions can be adjusted by reaction temperature and salt concentration. That is, higher stringency conditions are achieved at higher temperatures, and higher stringency conditions are achieved at lower salt concentrations. For example, when a probe having a length of 50 to 75 bases is used, higher stringency conditions can be obtained by setting the hybridization conditions to 40 to 44 ° C., 0.2% SDS, and 6 × SSC.
　また、プローブと標識を有するゲノムDNA断片とのハイブリダイズは、標識に基づいて検出することができる。すなわち、上述した標識を有するゲノムDNA断片とプローブのハイブリダイズ反応の後、未反応のゲノムDNA断片等を洗浄し、その後、プローブに対して特異的にハイブリダイズしたゲノムDNA断片の標識を観察する。例えば、標識が蛍光物質である場合にはその蛍光波長を検出し、標識が色素分子であればその色素波長を検出する。より具体的には、通常のDNAマイクロアレイ解析に使用している、蛍光検出装置やイメージアナライザー等の装置を使用することができる。 Further, hybridization between the probe and the genomic DNA fragment having a label can be detected based on the label. That is, after the hybridization reaction of the genomic DNA fragment having the above-described label and the probe, the unreacted genomic DNA fragment is washed, and then the label of the genomic DNA fragment specifically hybridized to the probe is observed. . For example, when the label is a fluorescent substance, the fluorescence wavelength is detected, and when the label is a dye molecule, the dye wavelength is detected. More specifically, an apparatus such as a fluorescence detection apparatus or an image analyzer that is used for normal DNA microarray analysis can be used.
　以上のように、核酸増幅法を利用する方法或いはDNAマイクロアレイを使用する方法により、供試イチゴ属植物が上述したイチゴ属植物うどんこ病抵抗性関連マーカーを有するか否か判断することができる。ここで、上述したように、イチゴ属植物うどんこ病抵抗性関連マーカーはうどんこ病抵抗性に優れるという形質に連鎖するため、イチゴ属植物うどんこ病抵抗性関連マーカーが存在していれば、うどんこ病抵抗性に優れる品種・系統と判断できる。 As described above, whether or not the strawberry genus plant has the aforementioned strawberry plant powdery mildew resistance-related marker can be determined by a method using a nucleic acid amplification method or a method using a DNA microarray. Here, as described above, the strawberry genus plant powdery mildew resistance-related marker is linked to the trait that it is excellent in powdery mildew resistance, so if the strawberry genus plant powdery mildew resistance-related marker exists, It can be judged that it is a variety and strain excellent in powdery mildew resistance.
　特に、上述した方法では、供試イチゴ属植物を実際のうどんこ病抵抗性試験を実施可能な程度まで成長させる必要はなく、例えば後代系統の種子や当該種子を発芽させた幼苗を使用することができる。したがって、イチゴ属植物うどんこ病抵抗性関連マーカーを利用することによって、供試イチゴ属植物を生育させるための圃場やその他、生育のためのコストを大幅に削減することができる。また、イチゴ属植物うどんこ病抵抗性関連マーカーを利用することによって、実際にうどんこ菌の原因微生物（Sphaerotheca aphanis）を感染させる必要がなく、大規模専用温室や専用圃場、外部との隔離施設など設備等にかかるコストを削減できる。 In particular, in the above-described method, it is not necessary to grow the test strawberry genus plant to such an extent that an actual powdery mildew resistance test can be carried out. For example, use seeds of progeny lines or seedlings that germinate the seeds. Can do. Therefore, by using the strawberry genus plant powdery mildew resistance-related marker, the field for growing the strawberry genus plant and other costs for growth can be greatly reduced. In addition, by using a marker related to resistance to powdery mildew in the genus Strawberry, there is no need to actually infect the microorganism causing causative of powdery mildew (Sphaerotheca aphanis), a large-scale dedicated greenhouse, a dedicated field, and an external isolation facility The cost for facilities etc. can be reduced.
　特に、イチゴ属植物の新品種作出に際して、先ず、交配により数万種類の交配種を作製した後、実生選抜に先立って若しくは実生選抜に代えて、イチゴ属植物うどんこ病抵抗性関連マーカーを利用した判断を行うことが好ましい。これにより、実際の圃場において栽培する個体数を大幅に削減することができ、イチゴ属植物の新品種作出に係る手間やコストを大幅に削減することができる。 In particular, when producing new varieties of Strawberry genus plants, first, tens of thousands of hybrids are prepared by crossing, and then a strawberry genus plant powdery mildew resistance-related marker is used prior to seedling selection or instead of seedling selection. It is preferable to make the above judgment. Thereby, the number of individuals cultivated in an actual field can be significantly reduced, and the labor and cost for producing a new variety of strawberry genus plants can be greatly reduced.
　或いは、イチゴ属植物の新品種作出に際して、先ず、交配に使用する親品種におけるイチゴ属植物うどんこ病抵抗性関連マーカーの存在の有無を判定し、うどんこ病抵抗性に優れた親品種を選抜することもできる。うどんこ病抵抗性に優れた親品種を優先的に使用して後代系統を作出することで、うどんこ病抵抗性に優れた後代系統が高頻度に出現すると期待できる。これにより、優良な系統を栽培する数を大幅に削減することができ、イチゴ属植物の新品種作出に係る手間やコストを大幅に抑制することができる。 Alternatively, when creating a new variety of Strawberry genus plants, first determine the presence or absence of a strawberry genus plant powdery mildew resistance-related marker in the parent varieties used for mating, and select the parent varieties with excellent powdery mildew resistance. You can also It is expected that progeny lines excellent in powdery mildew resistance will appear frequently by creating a progeny line preferentially using parent varieties excellent in powdery mildew resistance. Thereby, the number which cultivates an excellent system | strain can be reduced significantly, and the effort and cost which concern on the new variety production of a strawberry genus plant can be suppressed significantly.
　以下、実施例により本発明をより詳細に説明するが、本発明の技術的範囲は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail by way of examples. However, the technical scope of the present invention is not limited to the following examples.
１．DNAマイクロアレイ用プローブの作成
(1)材料
　イチゴ品種「みやざきなつはるか」と「０８と－ｆ」を用いた。
(2)制限酵素処理 
　これらイチゴ品種それぞれについて、Dneasy Plant Mini kit（QIAGEN社）を使用してゲノムDNAを抽出した。抽出したゲノムDNA(150ng)を制限酵素PstI(NEB社、5unit)で37℃、1時間処理した。
(3)アダプターライゲーション 
　(2)で処理したゲノムDNA断片(150ng)にPstI配列アダプター（5’-CACGATGGATCCAGTGCA-3’（配列番号２０）、5’-CTGGATCCATCGTGCA-3’（配列番号２１））とT4 DNA Ligase（NEB社、200 unit）を加え、16℃で1時間、その後55℃で20分間、その後37℃で30分間の条件でライゲーション反応を行った。次に、処理したサンプルに制限酵素BstNI（NEB社、6 unit）を添加、60℃で1時間処理した。
(4)PCR増幅
　(3)で得られた、BstNI処理後のサンプル（15ng）にPstI配列アダプター認識プライマー（5’-GATGGATCCAGTGCAG-3’（配列番号２２））とTaq polymerase（タカラバイオ社、PrimeSTAR、1.25unit）を加え、PCR（98℃を10秒間、55℃を15秒間、72℃を1分間、30サイクル後、72℃で3分間処理後、4℃で保存）でDNA断片を増幅した。
(5)ゲノムシークエンス取得
　(4)においてPCR増幅したゲノムDNA断片について、Hiseq2000、Miseq（Illumina社）により塩基配列情報を取得した。
(6)プローブ設計及びDNAマイクロアレイの作成
　(5)のゲノムシークエンス情報をもとに50～60bpのプローブを設計した。設計したプローブの塩基配列情報をもとに、これらプローブを有するDNAマイクロアレイを作製した。 1. Creating DNA microarray probes
(1) Material Strawberry varieties “Miyazaki Natsuharu” and “08 and -f” were used.
(2) Restriction enzyme treatment
For each of these strawberry cultivars, genomic DNA was extracted using Dneasy Plant Mini kit (QIAGEN). The extracted genomic DNA (150 ng) was treated with the restriction enzyme PstI (NEB, 5 units) at 37 ° C. for 1 hour.
(3) Adapter ligation
The PstI sequence adapter (5'-CACGATGGATCCAGTGCA-3 '(SEQ ID NO: 20), 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 21)) and T4 DNA Ligase (NEB) were added to the genomic DNA fragment (150 ng) treated in (2). , 200 units) was added, and the ligation reaction was performed at 16 ° C. for 1 hour, then at 55 ° C. for 20 minutes, and then at 37 ° C. for 30 minutes. Next, restriction enzyme BstNI (NEB, 6 units) was added to the treated sample and treated at 60 ° C. for 1 hour.
(4) PCR amplification (3), BstNI-treated sample (15 ng), PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3 '(SEQ ID NO: 22)) and Taq polymerase (Takara Bio, PrimeSTAR) 1.25unit) and amplified DNA fragments by PCR (98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 1 minute, 30 cycles, treated at 72 ° C for 3 minutes, and stored at 4 ° C) .
(5) Genome sequence acquisition For the genomic DNA fragment PCR-amplified in (4), base sequence information was acquired by Hiseq 2000 and Miseq (Illumina).
(6) Probe design and preparation of DNA microarray Based on the genomic sequence information of (5), a probe of 50-60 bp was designed. Based on the nucleotide sequence information of the designed probes, a DNA microarray having these probes was prepared.
２．シグナルデータ取得
(1)材料
　イチゴ品種「みやざきなつはるか」、「０８と－ｆ」及びこれらの交雑後代147系統を用いた。
(2)制限酵素処理
　これらイチゴ品種及び交雑後代からそれぞれゲノムDNAをDneasy Plant Mini kit（QIAGEN社）により抽出した。抽出したゲノムDNA(150ng)を制限酵素PstI(NEB社、6unit)で37℃、1時間処理した。
(3)アダプターライゲーション
　(2)で処理したゲノムDNA断片(150ng)にPstI配列アダプター(5’-CACGATGGATCCAGTGCA-3’（配列番号２０）、5’-CTGGATCCATCGTGCA-3’ （配列番号２１）)とT4 DNA Ligase(NEB社、200 unit)を加え、16℃で1時間、その後55℃で20分間、その後37℃で30分間の条件でライゲーション反応を行った。次に、処理したサンプルに制限酵素BstNI（NEB社、6 unit）を添加、60℃で1時間処理した。
(4)PCR増幅
　(3)で得られたBstNI処理後のサンプル(15ng)にPstI配列アダプター認識プライマー(5’-GATGGATCCAGTGCAG-3’（配列番号２２）)とTaq polymerase(タカラバイオ社PrimeSTAR、1.25unit)を加え、PCR(98℃を10秒間、55℃を15秒間、72℃を1分間を1サイクルとして30サイクル実施後、72℃で3分間処理後、4℃で保存)でゲノムDNA断片を増幅した。
(5)ラベル化
　上述した(4)で増幅したDNA断片をカラム（QIAGEN社）で精製後、 NimbleGen Arrays User’s Guideに従い、NimbleGen One-Color DNA Labeling Kit（ロシュ・ダイアグノステックス社）を用いラベル化した。
(6)ハイブリ・シグナル検出 
　(5)のラベル化サンプルと、上記１．で作製したDNAマイクロアレイとを用いて、アジレントIn-situ オリゴDNAマイクロアレイ キット アレイCGH(aCGH)法に従いハイブリ処理をし、各サンプルのシグナルを検出した。 2. Signal data acquisition
(1) Materials Strawberry varieties “Miyazaki Natsuharu”, “08 and -f”, and 147 progenies of these hybrids were used.
(2) Restriction enzyme treatment Genomic DNA was extracted from these strawberry cultivars and hybrid progenies using the Dneasy Plant Mini kit (QIAGEN). The extracted genomic DNA (150 ng) was treated with the restriction enzyme PstI (NEB, 6unit) at 37 ° C. for 1 hour.
(3) Adapter ligation The genomic DNA fragment (150 ng) treated in (2) was added to the PstI sequence adapter (5'-CACGATGGATCCAGTGCA-3 '(SEQ ID NO: 20), 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 21)) and T4. DNA Ligase (NEB, 200 unit) was added, and a ligation reaction was performed at 16 ° C. for 1 hour, then at 55 ° C. for 20 minutes, and then at 37 ° C. for 30 minutes. Next, restriction enzyme BstNI (NEB, 6 units) was added to the treated sample and treated at 60 ° C. for 1 hour.
(4) PCR amplification The sample (15 ng) after BstNI treatment obtained in (3) was subjected to PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3 '(SEQ ID NO: 22)) and Taq polymerase (Takara Bio PrimeSTAR, 1.25 unit), followed by PCR (98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 1 minute for 30 cycles, treated at 72 ° C for 3 minutes, and stored at 4 ° C). Was amplified.
(5) Labeling After purifying the DNA fragment amplified in (4) above with a column (QIAGEN), label using the NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics) according to the NimbleGen Arrays User's Guide. did.
(6) Hybrid signal detection
The labeled sample of (5) and the above 1. Using the DNA microarray prepared in Step 1, hybridization was performed according to the Agilent In-situ Oligo DNA Microarray Kit Array CGH (aCGH) method, and the signal of each sample was detected.
３．イチゴうどんこ病抵抗性のQTLの同定、および選抜マーカーの選定
(1)遺伝地図データシート作成
　「みやざきなつはるか」と「０８と－ｆ」との交雑後代147系統のシグナルデータから、「みやざきなつはるか」型の8,218マーカー及び「０８と－ｆ」型の8,039マーカーの遺伝子型データを取得した。遺伝子型データをもとに、遺伝地図作成ソフトAntMap (（Iwata H, Ninomiya S 2006） AntMap: constructing genetic linkage maps using an ant colony optimization algorithm. Breed Sci 56: 371-378）を用い、遺伝距離計算式Kosambiによりマーカーの遺伝地図データを取得した。
(2)イチゴうどんこ病表現型データの取得
　「みやざきなつはるか」と「０８と－ｆ」との交雑後代種子をハウス内で育苗し（147系統）、次年度の春季に野外圃場に定植して、夏季にイチゴうどんこ病の発症程度を調査した（図３）。罹病性個体については、微、中、激の3段階で、発症程度を評価した。 3. Identification of QTL for strawberry powdery mildew resistance and selection of selection markers
(1) Creation of genetic map data sheet Based on signal data of 147 lines of the progeny of “Miyazaki Natsuka Haruka” and “08 and -f”, “Miyazaki Natsuka Haruka” type 8,218 markers and “08 and -f” type 8,039 Marker genotype data was acquired. Based on the genotype data, genetic map calculation software AntMap ((Iwata H, Ninomiya S 2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm. Breed Sci 56: 371-378) The genetic map data of the marker was acquired by Kosambi.
(2) Acquisition of strawberry powdery mildew phenotype data The progeny seeds of “Miyazaki Natsuka Haruka” and “08 and -f” were raised in the house (147 lines) and planted in the field in the spring of the following year. In the summer, the degree of onset of strawberry powdery mildew was investigated (Fig. 3). For diseased individuals, the degree of onset was evaluated in three stages: fine, medium and intense.
　なお、本例で使用したうどんこ病菌は、岩手県盛岡市に土着している菌を自然感染させた。
(3)量的形質 （quantitative trait loci：QTL）の解析
　上記(1)で得られた遺伝地図データ及び上記(2)で得られたうどんこ病検定試験データ（うどんこ病発症程度）をもとに、遺伝解析ソフトQTL Cartographer （Wang S., C. J. Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC）を使い、Composite interval mapping （CIM）法により、QTL解析を行った。LODの閾値は2.5を用いた。その結果、「08と-f」の第１連鎖群のマーカーIB535110からIB726514の区間内にLOD値7.3のイチゴうどんこ病抵抗性に関する遺伝子の存在を確認した（表２、図４）。
(4)選抜マーカーの選定
　第１連鎖群の0cM～6.83cMの区間にあるイチゴうどんこ病抵抗性遺伝子領域周辺のマーカーを選抜マーカーとして選定した（図４、表１）。 In addition, the powdery mildew used in this example naturally infected the bacteria indigenous to Morioka City, Iwate Prefecture.
(3) Analysis of quantitative trait loci (QTL) The genetic map data obtained in (1) above and the powdery mildew test data (degree of powdery mildew onset) obtained in (2) above And QTL Cartographer (Wang S., CJ Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC) QTL analysis was performed by the mapping (CIM) method. The LOD threshold was 2.5. As a result, the presence of a gene related to strawberry powdery mildew resistance having a LOD value of 7.3 was confirmed in the section between markers IB535110 to IB726514 in the first linkage group of “08 and -f” (Table 2, FIG. 4).
(4) Selection of selection marker A marker around the strawberry powdery mildew resistance gene region in the 0cM to 6.83cM section of the first linkage group was selected as a selection marker (Fig. 4, Table 1).
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　なお、表２において効果の欄は、本QTLが、うどんこ病発症程度（無0、微1、中2、激3）に及ぼす影響をあらわしている。すなわち、効果の数値が負である場合、うどんこ病発症程度を小さくする方向に作用し、当該QTLはうどんこ病抵抗性が向上する形質に連鎖することを意味している。 In Table 2, the effect column shows the effect of this QTL on the incidence of powdery mildew (none, 0, 1, 2, 3). That is, when the numerical value of the effect is negative, it acts to reduce the onset of powdery mildew, meaning that the QTL is linked to a trait that improves powdery mildew resistance.
　そして、図４に示すように、当該ピークの近傍に位置するマーカーは、うどんこ病抵抗性を向上させる機能を有する原因遺伝子（群）と連鎖して遺伝するため、イチゴ属植物うどんこ病抵抗性関連マーカーとして使用できることが示された。すなわち、図４に示した１９種類のマーカーは、イチゴ属植物うどんこ病抗性関連マーカーとして使用できることが明らかとなった。 And as shown in FIG. 4, since the marker located in the vicinity of the said peak is inherited in linkage with the causative gene (group) which has the function to improve powdery mildew resistance, strawberry genus plant powdery mildew resistance. It has been shown that it can be used as a sex-related marker. That is, it was clarified that the 19 types of markers shown in FIG. 4 can be used as markers associated with powdery mildew genus plant.
４．未知系統の選抜
(1)イチゴうどんこ病表現型データの取得
　上記３．(2)イチゴうどんこ病表現型データの取得で記載した系統とは別に、「みやざきなつはるか」と「０８と－ｆ」との交雑後代種子をハウス内で育苗し（50系統：以降Ａ集団とする）、秋季に野外圃場に定植して、翌年の夏季にイチゴうどんこ病の発症程度を調査した。また、「みやざきなつはるか」と「おおきみ」との交雑後代種子（42系統：以降Ｂ集団とする）、及び「みやざきなつはるか」と「09s E-b 45e」との交雑後代種子（42系統：以降Ｅ集団とする）についても同様に育苗、定植し、うどんこ病発症程度を調査した（図５－１及び５－２）。
(2)ゲノムDNAの抽出
　新たに、イチゴ品種「みやざきなつはるか」、「０８と－ｆ」及びＡ集団について、Dneasy Plant Mini kit（QIAGEN社）によりゲノムDNAを抽出した。
(3)制限酵素処理及びアダプターライゲーション
　抽出したゲノムDNA（150ng）を制限酵素PstI（NEB社、5unit）で37℃、1時間処理後、PstI処理したサンプルにPstI配列アダプター(5’-CACGATGGATCCAGTGCA-3’（配列番号２０）、5’-CTGGATCCATCGTGCA-3’（配列番号２１）)とT4 DNA Ligase（NEB社、200 unit）を加え、16℃で1時間処理後、55℃で20分、その後、37℃で30分処理した。処理したサンプルに制限酵素BstNI（NEB社、6 unit）を添加、60℃で1時間処理した。
(4)DNA断片の増幅
　上記(3)にてBstNI処理したサンプル（15ng）にPstI配列アダプター認識プライマー（5’-GATGGATCCAGTGCAG-3’（配列番号２２））とTaq polymerase（タカラバイオ社、PrimeSTAR、1.25 unit）を加え、PCR(98℃を10秒間、55℃を15秒間、72℃を1分間を1サイクルとして30サイクル実施後、72℃で3分間処理後、4℃で保存)でDNA断片を増幅した。
(5)ラベル化
　上記(4)にて増幅したDNA断片をカラム（QIAGEN社）で精製後、 NimbleGen Arrays User’s Guideに従い、NimbleGen One-Color DNA Labeling Kit（ロシュ・ダイアグノステックス社）を用いラベル化した。
(6)ハイブリ・シグナル検出
　上記(6)で蛍光標識したサンプルと上記１．で作成したアレイを用い、アジレントIn-situ オリゴDNAマイクロアレイ キット アレイCGH (aCGH) 法に従いハイブリ処理をし、各サンプルのシグナルを検出した。
(7)選定した選抜マーカーの検定
　上記Ａ集団中で、イチゴうどんこ病抵抗性遺伝子領域周辺のマーカーを選定し（表１）、選抜マーカーとしたものでアレイシグナル値とＡ集団の表現型とを比較すると90.0%～98.0%一致していた（図６－１～６－５）。なお、図６－１～６－５において、高いアレイシグナル値には下線を付した。これらの結果から、表１に示したマーカーを利用することで、うどんこ病抵抗性に優れる系統、劣る系統の選抜が可能と考えられた。 4). Selection of unknown lines
(1) Acquisition of strawberry powdery mildew phenotype data 3. (2) Aside from the lines described in the acquisition of strawberry powdery mildew phenotype data, progeny seeds of hybrids of “Miyazaki Natsuka Haruka” and “08 and -f” were raised in the house (50 lines: group A thereafter) And planted in an outdoor field in the fall, and investigated the degree of onset of strawberry powdery mildew in the summer of the following year. In addition, progeny seeds of hybrids between “Miyazaki Natsuka Haruka” and “Ookimi” (42 lines: hereinafter referred to as Group B), and progeny seeds of “Miyazaki Natsuka Haruka” and “09s Eb 45e” (42 lines: subsequent) Similarly, for the E group, seedlings, planting, and the occurrence of powdery mildew were investigated (FIGS. 5-1 and 5-2).
(2) Extraction of genomic DNA Genomic DNA was newly extracted from the strawberry cultivars “Miyazaki Natsuka Haruka”, “08 and -f” and Group A using the Dneasy Plant Mini kit (QIAGEN).
(3) Restriction enzyme treatment and adapter ligation After the extracted genomic DNA (150 ng) was treated with restriction enzyme PstI (NEB, 5unit) at 37 ° C for 1 hour, the PstI sequence adapter (5'-CACGATGGATCCAGTGCA-3 '(SEQ ID NO: 20), 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 21)) and T4 DNA Ligase (NEB, 200 units) were added, treated at 16 ° C for 1 hour, then at 55 ° C for 20 minutes, Treated at 37 ° C. for 30 minutes. Restriction enzyme BstNI (NEB, 6 units) was added to the treated sample and treated at 60 ° C. for 1 hour.
(4) Amplification of DNA fragment A sample (15 ng) treated with BstNI in the above (3) was subjected to PstI sequence adapter recognition primer (5′-GATGGATCCAGTGCAG-3 ′ (SEQ ID NO: 22)) and Taq polymerase (Takara Bio Inc., PrimeSTAR, 1.25 unit) and DNA fragment by PCR (98 ° C for 10 seconds, 55 ° C for 15 seconds, 72 ° C for 1 minute for 30 cycles, treated at 72 ° C for 3 minutes, and stored at 4 ° C) Was amplified.
(5) Labeling After purifying the DNA fragment amplified in (4) above with a column (QIAGEN), labeling using the NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics) according to the NimbleGen Arrays User's Guide did.
(6) Hybrid signal detection The sample fluorescently labeled in (6) above and 1. Using the array prepared in step 1, hybridization was performed according to the Agilent In-situ Oligo DNA Microarray Kit Array CGH (aCGH) method, and the signal of each sample was detected.
(7) Selected marker marker test In the above A population, markers around the strawberry powdery mildew resistance gene region were selected (Table 1), and the selected marker was used as the array signal value and the phenotype of the A population. 90.0% to 98.0% (FIGS. 6-1 to 6-5). In FIGS. 6-1 to 6-5, high array signal values are underlined. From these results, it was considered that by using the markers shown in Table 1, it was possible to select strains with excellent and inferior powdery mildew resistance.
５．PCRベースマーカーよる選抜・検定１
(1)ゲノムDNAの抽出
　イチゴ品種「みやざきなつはるか」、「０８と－ｆ」、「おおきみ」、「09s E-b 45e」、Ａ集団（51系統、）Ｂ集団(42系統)及びＥ集団(42系統)について、Dneasy Plant Mini kit（QIAGEN社）によりゲノムDNAを抽出した。
(2)プライマー作製
　PCRプライマー解析ソフトPrimer3を使い、IB535110の配列情報（配列番号１）から、IB535110を識別するプライマーを作製した（35110_v1F： ACACATATATGAATCGGAGCCA（配列番号２３）、35110_v1R： GCTCAAGATGCTCAATCGAA（配列番号２４））。
(3)PCR増幅と選抜マーカーの検定
　交雑後代Ａ集団、Ｂ集団、Ｅ集団のゲノムDNA（15 ng）に、上記プライマー（35110_v1F及び35110_v1R）とTaq polymerase（タカラバイオ社、Tks Gflex DNA Polymerase、1.25 unit）を加え、PCR（94℃で1分間、98℃で10秒間、60℃で15秒間、68℃で30秒間を１サイクルとして30サイクル実施後、4℃で保存）で増幅した。PCR増幅したDNA断片は、TapeStation D1000（アジレント社）により確認した。Ａ集団、Ｂ集団及びＥ集団について行った結果を図７－１及び７－２、図８－１及び８－２並びに図９－１及び９－２にそれぞれ示した。なお、図７－１～図９－２において、Ｍのレーンは「みやざきなつはるか」を示し、Ｚのレーンは「０８と－ｆ」を示している。また、これらの結果を図１０－１及び１０－２にまとめて示した。なお、図１０－１及び１０－２において下線は、表現型とPCRベースマーカーの結果とが一致しなかった場合を示している。図７－１～図１０－２に示したように、バンドパターンと表現型の一致率は非常に高く（98.5%）、IB535110を特異的に増幅するプライマーを用いた核酸増幅方法により、うどんこ病抵抗性に優れる系統、劣る系統の選抜ができることが明らかとなった。 5. Selection / testing with PCR-based markers 1
(1) Genomic DNA extraction Strawberry varieties “Miyazaki Natsuka Haruka”, “08 and -f”, “Ookimi”, “09s Eb 45e”, Group A (51 lines), Group B (42 lines) and Group E ( For 42 lines), genomic DNA was extracted with Dneasy Plant Mini kit (QIAGEN).
(2) Primer preparation Primer 3 was used to prepare primers that identify IB535110 from the sequence information (SEQ ID NO: 1) of IB535110 (35110_v1F: ACACATATATGAATCGGAGCCA (SEQ ID NO: 23), 35110_v1R: GCTCAAGATGCTCAATCGAA (SEQ ID NO: 24) ).
(3) PCR amplification and selection marker test The genomic DNA (15 ng) of the progeny A, B, and E of the hybrid was added to the primers (35110_v1F and 35110_v1R) and Taq polymerase (Takara Bio Inc., Tks Gflex DNA Polymerase, 1.25 unit) was added and amplified by PCR (30 cycles of 94 ° C for 1 minute, 98 ° C for 10 seconds, 60 ° C for 15 seconds, 68 ° C for 30 seconds, then stored at 4 ° C). The PCR amplified DNA fragment was confirmed by TapeStation D1000 (Agilent). The results obtained for the A group, B group, and E group are shown in FIGS. 7-1 and 7-2, FIGS. 8-1 and 8-2, and FIGS. 9-1 and 9-2, respectively. In FIGS. 7-1 to 9-2, the M lane indicates “Miyazaki Natsuka Haruka”, and the Z lane indicates “08 and −f”. These results are collectively shown in FIGS. 10-1 and 10-2. In FIGS. 10-1 and 10-2, the underline indicates the case where the phenotype does not match the PCR base marker result. As shown in FIGS. 7-1 to 10-2, the coincidence ratio between the band pattern and the phenotype is very high (98.5%), and the noodles are amplified by a nucleic acid amplification method using a primer that specifically amplifies IB535110. It became clear that it was possible to select strains with excellent disease resistance and inferior strains.
６．PCRベースマーカーよる選抜・検定２
(1)ゲノムDNAの抽出
　イチゴ品種「みやざきなつはるか」、「０８と－ｆ」、「おおきみ」、「09s E-b 45e」、Ａ集団（51系統、）Ｂ集団(42系統)及びＥ集団(42系統)について、Dneasy Plant Mini kit（QIAGEN社）によりゲノムDNAを抽出した。
(2)プライマー作製
　PCRプライマー解析ソフトPrimer3を使い、IB522828の配列情報（配列番号２）から、 IB522828を識別するプライマーを作製した（22828_v6F：CTTTGACGCCTACTGCATTA （配列番号２５）、22828_v6R：GGTTGGGCTTCGTTAAATCT（配列番号２６））。
(3)PCR増幅と選抜マーカーの検定
　交雑後代Ａ集団、Ｂ集団、Ｅ集団のゲノムDNA（15 ng）に、上記プライマー（22828_v6F及び22828_v6R）とTaq polymerase（タカラバイオ社、Tks Gflex DNA Polymerase、1.25 unit）を加え、PCR（94℃で1分間、98℃で10秒間、60℃で15秒間、68℃で30秒間を１サイクルとして30サイクル実施後、4℃で保存）で増幅した。PCR増幅したDNA断片は、TapeStation D1000（アジレント社）により確認した。Ａ集団、Ｂ集団及びＥ集団について行った結果を図１１－１～１１－３、図１２－１及び１２－２並びに図１３－１及び１３－２にそれぞれ示した。なお、図１１－１～図１３－２において、Ｍのレーンは「みやざきなつはるか」を示し、Ｚのレーンは「０８と－ｆ」を示し、Ｏのレーンは「おおきみ」を示している。また、これらの結果を図１４－１及び１４－２にまとめて示した。なお、図１４－１及び１４－２において下線は、表現型とPCRベースマーカーの結果とが一致しなかった場合を示している。図１１－１～図１４－２に示したように、バンドパターンと表現型の一致率は非常に高く（98.5%）、IB522828を特異的に増幅するプライマーを用いた核酸増幅方法により、うどんこ病抵抗性に優れる系統、劣る系統の選抜ができることが明らかとなった。 6). Selection / testing 2 with PCR base markers
(1) Genomic DNA extraction Strawberry varieties “Miyazaki Natsuka Haruka”, “08 and -f”, “Ookimi”, “09s Eb 45e”, Group A (51 lines), Group B (42 lines) and Group E ( For 42 lines), genomic DNA was extracted with Dneasy Plant Mini kit (QIAGEN).
(2) Primer preparation Primer 3 was used to prepare a primer for identifying IB522828 from the sequence information (SEQ ID NO: 2) of IB522828 (22828_v6F: CTTTGACGCCTACTGCATTA (SEQ ID NO: 25), 22828_v6R: GGTTGGGCTTCGTTAAATCT (SEQ ID NO: 26) ).
(3) PCR amplification and selection marker test The genomic DNA (15 ng) of the progeny A, B, and E of the hybrid was mixed with the above primers (22828_v6F and 22828_v6R) and Taq polymerase (Takara Bio Inc., Tks Gflex DNA Polymerase, 1.25 unit) was added and amplified by PCR (30 cycles of 94 ° C for 1 minute, 98 ° C for 10 seconds, 60 ° C for 15 seconds, 68 ° C for 30 seconds, then stored at 4 ° C). The PCR amplified DNA fragment was confirmed by TapeStation D1000 (Agilent). The results obtained for the A group, B group and E group are shown in FIGS. 11-1 to 11-3, FIGS. 12-1 and 12-2, and FIGS. 13-1 and 13-2, respectively. In FIGS. 11-1 to 13-2, the M lane indicates “Miyazaki Natsuka Haruka”, the Z lane indicates “08 and −f”, and the O lane indicates “Ohmi”. . These results are collectively shown in FIGS. 14-1 and 14-2. In FIGS. 14-1 and 14-2, the underline indicates a case where the phenotype does not match the result of the PCR base marker. As shown in FIGS. 11-1 to 14-2, the coincidence rate between the band pattern and the phenotype is very high (98.5%), and by using the nucleic acid amplification method using a primer that specifically amplifies IB522828, udon It became clear that it was possible to select strains with excellent disease resistance and inferior strains.
　本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into this specification as they are.

Claims (7)

  1. 　イチゴ属植物の染色体における配列番号１に示す塩基配列及び配列番号１９に示す塩基配列により挟まれる連続する核酸領域からなる、イチゴ属植物うどんこ病抵抗性関連マーカー。 A strawberry genus plant powdery mildew resistance-related marker comprising a continuous nucleic acid region sandwiched between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 19 in the chromosome of a strawberry genus plant.
  2. 　上記核酸領域は、配列番号１～１９からなる群から選ばれるいずれか１の塩基配列又は当該塩基配列の一部を含むことを特徴とする請求項１記載のイチゴ属植物うどんこ病抵抗性関連マーカー。 The strawberry genus plant powdery mildew resistance-related disease according to claim 1, wherein the nucleic acid region comprises any one of a base sequence selected from the group consisting of SEQ ID NOs: 1 to 19 or a part of the base sequence. marker.
  3. 　上記核酸領域は、イチゴ属植物の染色体における配列番号１に示す塩基配列と配列番号７に示す塩基配列とにより挟み込まれる領域に位置することを特徴とする請求項１記載のイチゴ属植物うどんこ病抵抗性関連マーカー。 The strawberry genus plant powdery mildew according to claim 1, wherein the nucleic acid region is located in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 7 in the chromosome of the strawberry genus plant. Resistance-related marker.
  4. 　少なくとも一方の親がイチゴ属植物である後代植物の染色体及び/又は当該親のイチゴ属植物の染色体を抽出する工程と、
    　上記で得られた染色体における請求項１乃至３いずれか一項記載のイチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を判定する工程とを含む、うどんこ病抵抗性が向上したイチゴ属植物系統の製造方法。     Extracting a chromosome of a progeny plant and / or a chromosome of the parent strawberry genus plant wherein at least one parent is a strawberry genus plant; and
    A strawberry having improved powdery mildew resistance, comprising the step of determining the presence / absence of the marker associated with powdery mildew plant resistance resistance according to any one of claims 1 to 3 in the chromosome obtained above. A method for producing a genus plant line.
  5. 　上記判定する工程では、上記イチゴ属植物うどんこ病抵抗性関連マーカーを特異的に増幅するプライマーを用いた核酸増幅反応により当該イチゴ属植物うどんこ病抵抗性関連マーカーの存在・非存在を判定することを特徴とする請求項４記載のイチゴ属植物系統の製造方法。 In the determining step, the presence or absence of the strawberry plant powdery mildew resistance-related marker is determined by a nucleic acid amplification reaction using a primer that specifically amplifies the strawberry plant powdery mildew resistance-related marker. The method for producing a strawberry genus plant line according to claim 4.
  6. 　上記判定する工程では、上記イチゴ属植物うどんこ病抵抗性関連マーカーに対応するプローブを備えるDNAチップを使用することを特徴とする請求項４記載のイチゴ属植物系統の製造方法。 The method for producing a strawberry genus plant line according to claim 4, wherein a DNA chip comprising a probe corresponding to the strawberry genus plant powdery mildew resistance-related marker is used in the determining step.
  7. 　上記後代植物は種子又は幼苗であり、当該種子又は幼苗から染色体を抽出することを特徴とする請求項４記載のイチゴ属植物系統の製造方法。 The method according to claim 4, wherein the progeny plant is a seed or a seedling, and a chromosome is extracted from the seed or the seedling.
PCT/JP2016/058711 2015-03-18 2016-03-18 Powdery mildew resistance-associated markers of fragaria genus plants and use thereof WO2016148279A1 (en)

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WO2015034040A1 (en) * 2013-09-09 2015-03-12 トヨタ自動車株式会社 Anthracnose resistance-associated marker for plant of genus fragaria, and use thereof

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