JP4469992B2 - Selection marker for clubroot resistant rapeseed - Google Patents

Description

  The present invention relates to a marker for selection of clubroot resistant rapeseed. More specifically, a marker useful for individual selection of rapeseed having a root-knot resistance trait, an oligonucleotide capable of specifically amplifying the marker, and a clubroot-resistant rapeseed using the marker and the oligonucleotide It relates to the selection method of the kind.

  Clubroot disease is a soil disease that causes severe damage to cruciferous plants in general, and is a disease that is extremely difficult to control. In plants that have developed clubroot, the roots become bumpy and cannot absorb enough nutrients, which causes developmental problems. In some cruciferous plants, resistant varieties are bred, but there are many cases where even resistant varieties are affected by race differentiation of the pathogen.

  Currently, countermeasures in areas with frequent clubroots rely on pesticide control, but it is difficult to completely suppress or kill the causative pathogens with this method. In addition, the use of a large amount of fungicide is feared to have an adverse effect on the environment due to residual components, and the production of agricultural products with reduced use of chemicals including fungicides is desired from the market.

  The rapeseed is an edible rapeseed belonging to Brassica napus L. (Rapeseed rapeseed), but there has been no varieties that have resistance to clubroot and are suitable for the fruit and vegetable market. Therefore, in order to obtain a variety that can overcome the disease, breeding of a new variety by crossing with a material having a root-knot resistance trait has been attempted. The feed turnip cultivar Rutabaga currently used as a material for crossing belongs to Brassica napus L. and is a cultivar with strong resistance that hardly causes disease, and its disease resistance traits are other It is different from rape. However, since the resistant material is feed turnips, the progenies of the hybrid are likely to inherit traits that are not suitable for the fruit and vegetable market, and it is predicted that it will take a long time to develop new varieties.

  In addition, the current resistance test and selection of resistant individuals are carried out by a method of selecting individuals showing resistance after inoculation with pathogenic bacteria. However, this determination requires a cultivation period of about 5 weeks after sowing, and after completion of the test, soil and root nodules containing a large amount of pathogenic bacteria are generated. Furthermore, when the temperature and soil moisture are low, the disease cannot be caused and the accuracy of the test is low, so the test result is subject to seasonal or annual constraints. For this reason, facilities such as an artificial weather room are required to increase the accuracy of the verification, which hinders the efficiency of selective breeding. Under such circumstances, it is desired to develop a method for stably and efficiently selecting individuals having clubroot resistance at the seedling stage.

  On the other hand, there is a selection method using DNA information as a trait marker. This method is a selection method that uses the property that DNA information does not change over the life of the organism, and can be easily affected by selection at the growth stage before the plant individual expresses the desired trait and environmental changes. Allows selection of traits. That is, this method does not generate soil contaminated with pathogenic bacteria as in the conventional testing method by inoculation with pathogenic bacteria, and enables stable trait selection without being restricted by facility or time constraints.

Conventionally, as a DNA marker for discriminating resistance to clubroot disease, a target for Chinese cabbage is known (for example, see Patent Document 1). However, DNA markers are specific to each trait, even within the same crop, and even for the same cruciferous plant, the Chinese cabbage marker can be used for other cruciferous vegetables (eg, rapeseed). Cannot be used.
JP-A-9-117284

  An object of the present invention is to obtain a DNA marker for stably and efficiently selecting individuals of rapeseed having root-knot resistance at an early stage of seedling raising, and using it to rapeseed-resistant rapeseed Is to establish a selection method.

  In solving the above-mentioned problems, the inventors tried to use Chinese cabbage DNA markers for rapeseed. Although both are the same cruciferous plants and the disease symptoms of the affected individuals are similar, the DNA markers of Chinese cabbage could not select individuals for clubroot resistant rapeseed. This is because the marker is specific for each trait even for each crop or even within the same crop.

  Therefore, the present inventor has developed a marker applicable to rapeseed. First, DNA polymorphism analysis of rapeseed is performed by PCR using a synthetic oligonucleotide having an arbitrary base sequence and a cyclosatellite primer that amplifies a specific microsatellite region, and is effective in selecting root-resistant rapeseed A new DNA marker was acquired. Furthermore, this DNA marker was improved and a synthetic oligonucleotide primer capable of amplifying a single DNA band was prepared. And it discovered that a clubroot resistant individual | organism | solid could be selected efficiently in the breeding process if this primer was used.

  That is, the present invention provides a marker for selecting root-knot-resistant rapeseed comprising a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3.

  The present invention also provides the marker amplification primer. The primer is designed as an oligonucleotide for specifically hybridizing to a polynucleotide containing the base sequence represented by SEQ ID NO: 3 and amplifying the sequence.

  As a suitable aspect of the said oligonucleotide, the oligonucleotide containing the base sequence shown by sequence number 1 or sequence number 2 can be mentioned. The oligonucleotide may be used either alone or both depending on the nucleic acid amplification method used. As a particularly preferred form, there can be mentioned a primer pair comprising two kinds of oligonucleotides each including the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

  The present invention also provides a method for selecting clubroot resistant rapeseed using the marker and oligonucleotide of the present invention. This method is performed by detecting the marker from RNA or DNA extracted from rapeseed. In a preferred embodiment, the detection includes a nucleic acid amplification step using the oligonucleotide or primer pair of the present invention.

For example, the method for selecting a clubroot resistant rape of the present invention comprises the following steps:
1) Performing a nucleic acid amplification reaction using the oligonucleotide or primer pair of the present invention using RNA or DNA extracted from rapeseed as a template;
2) A clubroot resistant rapeseed is selected by detecting an amplification product containing the nucleotide sequence represented by SEQ ID NO: 3 from the obtained nucleic acid amplification product.
In the method, the detection can be performed using, for example, electrophoretic analysis.

  According to the present invention, it is possible to easily identify and select a clubroot resistant individual of rapeseed. The present invention improves the selection accuracy in the breeding of new varieties resistant to clubroot of rapeseed, and contributes to a great reduction in labor and the shortening of the breeding period that have been required so far. Furthermore, if the present invention is applied and developed, it will be possible to develop new DNA markers and breed new varieties having traits important for agricultural production such as early and late characteristics.

Hereinafter, the present invention will be described in detail.
1. Breeding of clubroot resistant strains In obtaining a marker for selection of clubroot resistant rapeseed, first of all, a strain resistant to clubroot of rapeseed is created. Rape root-resistant strains are produced by immobilizing the root-knot-resistant trait by repeating crossing, selection, and self-breeding. For example, a club with a clubroot resistant to radish and a rape susceptibility to clubroot is performed as parents, and a resistant line is selected from the progeny. Furthermore, by cultivating this resistant strain and fixing the resistance, it is possible to obtain rapeseed resistant to clubroot. As the clubroot resistant turnip, for example, a feed turnip variety rutabaga can be used. Rutabaga belongs to Brassica napus L. and is a cultivar with strong resistance that hardly causes clubroot disease, and its disease resistance traits are very strong unlike other rapeseed.

2. Marker for selection of root-knot-resistant rapeseed 2.1 Polymorphism analysis From the rapeseed-resistant strain of rapeseed obtained as described in the previous section, a DNA sequence that can serve as a marker for clubroot-resistant disease is searched. . As the clubroot resistance identification marker, it is preferable to use a DNA polymorphism that specifically appears only in the DNA of the clubroot resistant strain. Examples of such DNA polymorphism include RAPD and microsatellite polymorphism described below.

  RAPD (Random Amplified Polymorphic DNA) is a DNA polymorphism that is analyzed by PCR using a synthetic oligonucleotide with a sequence of 10 to 12 bases, and can be easily changed by changing the type of synthetic oligonucleotide used. And many can be obtained. The band showing this DNA polymorphism may itself be a DNA marker.

  A short repetitive base sequence in the genome of an organism is called a microsatellite and is known to be a stable individual identification marker because of its rich polymorphism. Such polymorphism is called microsatellite polymorphism. Several research institutions have developed microsatellite markers for each crop and primers for the markers. By using these markers, DNA polymorphisms can be obtained in almost the same manner as RAPD. Information on the microsatellite marker is, for example, S. Kresovich AKSzewc-McFadden SMBliek, USDA-ARS, Theor Appl Gene (1995) 91: 206-211, “Abundance and characterization of simple-sequence repeats (SSRs) isolated from a size-fractionated genomic library of Brassica napus L. (rapeseed) ”, CALLUM J. BELL AND JOSEPH R. ECKER, Plant Science Institute of Biology, University of Pennsylvania, Genomics 19,137-144 (1994),“ Assignment of 30 Microsatellite Loci to It can be obtained from the Linkage Map of Arabidopsis ”.

  In any of the above methods, DNA polymorphisms in various organisms can be easily known by performing PCR using DNA extracted from the target organism as a template and analyzing the amplified DNA. Therefore, it is used for genetic analysis, cultivar / individual identification, and F1 seed purity test.

  However, in the case of RAPD analysis, multiple DNA polymorphisms may be amplified in addition to the desired DNA polymorphism band, and the reproducibility of the results may be poor. It may not be suitable for the scene. Therefore, the DNA polymorphic fragment obtained is cloned, the nucleotide sequence is determined, and a primer that can amplify only the desired DNA polymorphism is designed and provided from this nucleotide sequence information (STS), resulting in stable results. It is necessary to obtain. On the other hand, microsatellite polymorphism is highly reproducible, so that redesign of primers is not necessary.

  In both RAPD analysis and microsatellite analysis, the synthetic oligonucleotide primer used can be easily synthesized using a commercially available DNA synthesizer.

2.2 Selection of markers for selection of root-knot-resistant rapeseed DNA polymorphisms effective as markers for selection of root-knot-resistant rapeseed are resistant strains and susceptible rapeseed in RAPD analysis or microsatellite analysis. And amplifying products that specifically appear only in the resistant strain can be obtained. Thus, amplification products (DNA bands) from several primers are selected as candidates for markers for selecting clubroot resistant rapeseed.

  Next, among the selected candidates, an optimal marker for selecting a clubroot resistant rapeseed is further identified. The identification is performed by performing RAPD analysis and microsatellite analysis similar to the previous section in several types of resistant lines, and selecting DNA polymorphisms that are commonly expressed in the resistant lines.

  For example, a line with fixed resistance and a susceptible rapeseed are crossed again, and the first, second, and third generation of the hybrid are obtained by selfing. Analyzes the relationship between resistance results from the clubroot inoculation test using the third generation of hybrids and amplification products using the second generation of crosses.

  As a result, the inventors have found that the amplification product (about 650 bp) obtained using the RAPD method using the oligonucleotide primer (OPH-06: SEQ ID NO: 7) is optimal as a marker for selection of root-knot-resistant rapeseed. It was confirmed. The polymorphism contained in this amplified product is located in the vicinity of the clubroot resistance gene and is therefore considered to be inherited in linkage with the clubroot resistance trait. As will be described later, this amplified fragment was confirmed to have the base sequence shown in SEQ ID NO: 3. Therefore, the polynucleotide containing the sequence can be used as a marker for selection of clubroot resistant rapeseed. That is, on the rapeseed gene, a continuous region having a length of 649 to 800 bases, particularly 649 to 700 bases including the base sequence shown in SEQ ID NO: 3 is suitable as a marker for selection of clubroot resistant rapeseed. Available to:

  Further, the marker for selection of clubroot resistant rapeseed of the present invention may be appropriately modified. For example, labeling bases such as fluorescent dyes (FITC, ROC, etc.), enzymes, proteins, radioisotopes, chemiluminescent substances (eg, DNP), biotin, DIG (digoxigenin), and modified bases such as peptide nucleic acids. Use. The oligonucleotide of the present invention includes such a modified product. For convenience, the base sequence of the sequence listing is described as DNA. However, when the marker is RNA, the base symbol “t” in the sequence listing should be read as “u”.

3. Oligonucleotide for selection of rapeseed resistant to root-knot disease However, DNA markers obtained by the RAPD method have reproducibility and other problems as described above, so that only polymorphic DNA can be specifically amplified. It is preferable to redesign the primer. That is, it is necessary to design a primer that can more specifically amplify the identified clubroot resistance marker.

  For this purpose, the amplification product by the OPH-06 primer is fractionated and purified, and then cloned to obtain a high-purity plasmid, and the base sequence of the marker portion is decoded using a DNA sequencer. It is necessary to design a marker-specific primer for selection of clubroot resistant rapeseed.

  Thus, the inventors have determined that the marker for selection of clubroot resistant rapeseed has the base sequence shown in SEQ ID NO: 3, hybridizes to the sequence, and can specifically amplify the primer. As a primer (1 set) having the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 was designed. The DNA fragment specifically amplified by this primer is a marker for selecting clubroot resistant rapeseed which is effective in selecting a clubroot resistant trait.

  It is not limited to the said primer, The oligonucleotide containing the base sequence shown by sequence number 1 or sequence number 2 can be used as a primer for selection of a root-knot-resistant rapeseed. The oligonucleotide may be used alone or as a pair of primer pairs depending on the nucleic acid amplification reaction used. The base length of the oligonucleotide is also determined according to the nucleic acid amplification reaction used. If it is a normal PCR reaction, it is 15-30 base length, Preferably it is 20-30 base length, Most preferably, it is 20-25 base length.

  The oligonucleotide of the present invention may be appropriately modified. For example, labeling bases such as fluorescent dyes (FITC, ROC, etc.), enzymes, proteins, radioisotopes, chemiluminescent substances (eg, DNP), biotin, DIG (digoxigenin), and modified bases such as peptide nucleic acids. Use. The oligonucleotide of the present invention includes such a modified product. For convenience, the base sequence of the sequence listing is described as DNA. However, when the oligonucleotide is RNA, the base symbol “t” in the sequence listing is read as “u”.

4). Selection and identification of clubroot resistant rapeseed Since the marker for selection of clubroot resistant rapeseed of the present invention is linked to the clubroot resistant gene of rapeseed, Can be used for detection. That is, the present invention provides a selection (or identification) method for clubroot-resistant rapeseed, which comprises detecting a marker for selection of clubroot-resistant rapeseed from RNA or DNA extracted from the rapeseed. provide.

  Here, detection of a marker for selection of root-knot-resistant rapeseed is a well-known method such as RT-PCR, dot blot, gene chip, nucleic acid hybridization using a solid phase nucleic acid sample such as a microarray or a membrane filter. Can be used arbitrarily.

  Usually, since the amount of marker sequence contained in RNA or DNA extracted from rapeseed is very small, it is preferable to amplify the marker region by performing a nucleic acid amplification reaction using the primer of the present invention. Then, by detecting the marker for selecting the clubroot of the present invention from the obtained amplification product, the individual resistant to clubroot of the rapeseed is selected. Here, the nucleic acid amplification reaction used is not particularly limited, and any nucleic acid amplification method such as SDA method, LCR method, LAMP method, ICAN method, NASBA method, etc. can be used in addition to the polymerase chain reaction (PCR).

  For example, a nucleic acid amplification reaction is performed using the oligonucleotide or primer pair of the present invention using RNA or DNA extracted from rapeseed as a template, and then the nucleotide sequence represented by SEQ ID NO: 3 is obtained from the obtained nucleic acid amplification product. By detecting the amplified product, the clubroot resistant rapeseed is selected. As the detection method, for example, electrophoresis analysis or the like can be used.

EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not restrict | limited by these.
Example 1: Establishment of a primer for selection of clubroot resistant rapeseed Breeding of root-knot-resistant rapeseed (Nabbana) Cross-cultivated root-knot-resistant varieties Rutabaga (feed turnips) and susceptible nabana were crossed, and resistant individuals were selected from their progenies by inoculation tests. This individual was bred with repeated inoculation tests to obtain a line with fixed resistance. After crossing this resistant strain with the susceptible nabana again to obtain the first generation of hybrids, the second generation and the third generation of hybrids were obtained by self-breeding.

2. RAPD analysis DNA was extracted from each generation of banana leaves according to a conventional method (cetyltrimethylammonium bromide (CTAB) method). In RAPD analysis, 200 μM dNTPs, 1.5 pmol RAPD primer and 0.2 unit Tth DNA polymerase (manufactured by Toyobo) were added using 10 ng of this DNA as a template to prepare a 10 μl reaction solution. As the RAPD primer, 792 kinds of commercially available random primers having 10 or 12 bases (Wako, Operon, Bex) were used. The PCR reactor was TP-3000 manufactured by Takara Corporation. After pretreatment at 94 ° C for 1 second, heat denaturation at 94 ° C for 1 second, annealing at 40 ° C for 2 minutes, and extension reaction at 72 ° C for 3 minutes. As a cycle, 45 cycles were repeated, and a temperature treatment at 72 ° C. for 7 minutes was further performed.

3. Microsatellite analysis In microsatellite analysis, using 10 ng of DNA as a template, 200 μM dNTPs, 2.5 pmol microsatellite primers 2 types [(locus) Bn6A2, Theor Appl Gene (1995) 91: 206-211; (locus) nga139, Genomics 19,137-144 ( 1994)] and 0.25 unit EX-Taq polymerase (manufactured by Takara) were added to prepare 10 μl of a reaction solution. The PCR reactor is TP-3000 manufactured by Takara, and after pretreatment at 94 ° C for 5 seconds, heat denaturation at 94 ° C for 5 seconds, annealing at 50-60 ° C for 30 seconds, and extension reaction at 72 ° C for 1 minute. One cycle was repeated 40 cycles, and a temperature treatment at 72 ° C. for 2 minutes was further performed.
Microsatellite primer:
Bn6A2 Forward: 5'-CTTTgTgTggACTTTTAgAACTTTA-3 '(SEQ ID NO: 4)
Bn6A2 Reverse: 5'-CgCAgCTTTTggCCCACCTg-3 '(SEQ ID NO: 5)
nga139 Forward: 5'-AgAgCTACCAgATCCgATgg-3 '(SEQ ID NO: 6)
nga139 Reverse: 5'-ggTTTCgTTTCACTATCCAgg-3 '(SEQ ID NO: 7)

  5-10 μl of PCR amplification product was electrophoresed in a 1.5% agarose gel containing 1 μg / ml ethidium bromide or a 1.5% agarose gel containing no ethidium bromide, followed by a buffer containing 1 μg / ml ethidium bromide The presence or absence of amplification products was analyzed by staining in a liquid and irradiating with ultraviolet rays.

4). Selection of markers for selection of root-knot-resistant rapeseed As a result, it was suggested that some synthetic nucleotide amplification products (DNA bands) might be effective for selection of root-knot-resistant rapeseed. Then, the relationship between the resistance by the clubroot inoculation test using the third generation of the cross and the amplification product using the second generation was analyzed. Among the PCR amplification products, those specific to the resistant parent rutabaga were searched, and among them, those specific to the progeny resistant strain were searched. Separation of resistance in the third generation of hybrids and separation of presence or absence of these searched PCR amplification products were examined by linkage and quantitative trait analysis. For linkage and quantitative trait analysis, MAPL (Plant Breeding Information Laboratory http://www.asahi-net.or.jp/~fh6y-uki/mapqtl.html; Yasuo Ukai et al. (1995) DNA polymorphism linkage This was done using the computer program MAPL for mapping and QTL analysis, see Breeding Journal 45: 139-142). As a result, it was found that the amplification product by the primer OPH-06 shown below and a band of about 650 bp are effective for selection of resistant rapeseed.
OPH-06 primer
OPH-06: 5'-ACGCATCGCA-3 '(SEQ ID NO: 8)

5). Next, PCR is performed using OPH-06 as a primer under the same conditions as in the previous section 2 to amplify the DNA fragment, which is fractionated and purified, and then cloned to extract a high-purity plasmid. did. The base sequence was decoded using a sequencer (ABI PRISM 310 Genetic Analyzer (PE Biosystems)). As a result, a DNA fragment having the sequence of SEQ ID NO: 3 was identified as a marker sequence for selection of clubroot resistant rapeseed. Therefore, a primer suitable for specific amplification of this fragment was designed, and one set of synthetic oligonucleotides having the following base sequences was determined.
Primers for selection of clubroot resistant rape:
Forward: 5'-CAGTCAAACCGAAGAAGAAG-3 '(SEQ ID NO: 1)
Reverse: 5'-ATGTTCAGTTTGTGCGATGC-3 '(SEQ ID NO: 2)

Example 2: Selection of a clubroot resistant strain A clubroot resistant variety rutabaga (feed turnip) and a susceptible rapeseed (nabanana) were crossed, and several resistant lines were obtained from the progeny. DNA was extracted in the same manner as in Example 1 from 6 lines (line 9, line 12, line 15, line 17, line 19, line 22, line 22) that self-bred and fixed the resistance trait. Using 10 ng of the extracted DNA as a template, 200 μM dNTPs, 4 pmol of selection primers (SEQ ID NOs: 1 and 2) determined in Example 1 and 0.5 unit EX-Taq polymerase (manufactured by Takara) were added to prepare 10 μl of a reaction solution. The PCR reactor is TP-3000 manufactured by Takara, and after pretreatment at 94 ° C for 3 seconds, heat denaturation at 94 ° C for 1 second, annealing at 60 ° C for 1 minute, and extension reaction at 72 ° C for 2 minutes. The cycle was repeated 35 cycles. The obtained amplification product was subjected to electrophoretic analysis according to Example 1.

  The results are shown in FIG. As is clear from FIG. 1, specific amplification of the marker for selection of the clubroot resistant rapeseed of the present invention was confirmed in the strong resistance lines 15, 17, and 19. Thus, the effectiveness of the marker and primer of the present invention was confirmed.

  By using the marker for selecting a clubroot resistant rapeseed of the present invention and the primer for the marker, it is possible to easily identify an individual of a rapeseed having root-knot resistance. Therefore, the present invention can be used for individual selection of clubroot resistant rapeseed at the early stage of seedling raising and breeding of clubroot resistant rapeseed.

SEQ ID NO: 1 Description of Artificial Sequence-Forward Primer for Selection of Clubroot Resistant Rapeseed
SEQ ID NO: 2 Description of artificial sequence-Reverse primer for selection of root-knot resistant rape
SEQ ID NO: 3 Description of artificial sequence-Marker for selection of clubroot resistant rapeseed SEQ ID NO: 4: Description of artificial sequence-Microsatellite primer (Bn6A2 Forward primer)
SEQ ID NO: 5: Description of artificial sequence-Microsatellite primer (Bn6A2 Reverse)
SEQ ID NO: 6: description of artificial sequence-primer for microsatellite (nga139 Forward)
SEQ ID NO: 7: Description of artificial sequence-Primer for microsatellite (nga139 Reverse)
SEQ ID NO: 8: description of artificial sequence-OPH-06 primer

1 is a photograph showing the results of electrophoresis of PCR amplification products of clubroot resistant rapeseed (Nabbana) strains in Example 2. FIG.

Claims (6)

  A marker for selection of root-knot-resistant rapeseed consisting of a polynucleotide comprising the base sequence represented by SEQ ID NO: 3. A primer pair comprising an oligonucleotide having a sequence consisting of nucleotides 29 to 48 of SEQ ID NO: 3 and an oligonucleotide having a sequence complementary to the sequence consisting of nucleotides 628 to 647 of SEQ ID NO: 3.   A method for selecting root-knot-resistant rapeseed, comprising detecting the marker according to claim 1 from RNA or DNA extracted from the rapeseed. The method according to claim 3, comprising performing a nucleic acid amplification reaction using the primer pair according to claim 2 or the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8 . A method for selecting clubroot resistant rapeseed comprising the following steps:
1) Using a RNA or DNA extracted from rapeseed as a template, a nucleic acid amplification reaction is performed using the primer pair according to claim 2 or the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 8 ;
2) A clubroot resistant rapeseed is selected by detecting an amplification product containing the nucleotide sequence represented by SEQ ID NO: 3 from the obtained nucleic acid amplification product.   The method according to claim 3, wherein the detection is performed using electrophoretic analysis.

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