JP2009278865A - Method for amplification of dna fragment - Google Patents

Abstract

Disclosed is a method for amplifying a DNA fragment that can selectively amplify only a labeled PCR product and has little individual difference in experimental results for the transposon display method of higher plants.
A method for amplifying a DNA fragment includes a DNA extraction step for extracting genomic DNA from cells of a higher plant, and an adapter addition for adding an adapter to the DNA fragment by partially digesting the genomic DNA with a restriction enzyme. Amplification was performed by the first PCR step and the first PCR step, which were performed by a one-side PCR method using a DNA fragment ligated with an adapter as a template and using only a primer specific for the transposon. A second PCR step for amplifying the DNA fragment by a double-sided PCR method using the DNA fragment as a template and a labeled primer specific to the transposon and a primer complementary to the adapter is included in this order.
[Selection] Figure 1

Description

  The present invention relates to a method for amplifying a DNA fragment, and more particularly, to a method for amplifying a DNA fragment used in a transposon display method for quickly and objectively associating a trait and a gene associated therewith.

  With the progress of molecular biology, in addition to animals such as humans and mice, the base sequences of the whole genome of higher plants have been analyzed. For example, the draft sequence of rice was also completed in 2002 by the International Rice Genome Sequencing Project (IRGSP).

  However, in order to create plants with high tolerance to environmental stresses such as drought and cold, and plants with high yields by genetic recombination, not only the entire base sequence of genomic DNA is determined, but also the genomic DNA described in The influence of each gene on plant traits, that is, the function of each gene must also be analyzed. One method for analyzing the function of a gene is the transposon display method described below.

  Here, the transposon is also called a transposable genetic element or a mobile genetic element (mobile genetic element, movable genetic element), and is randomly transferred from one site on the chromosomal DNA to another site. It is a DNA fragment having the property of translocation, and is known to cause gene disruption by being inserted into the translocation destination gene. For transposons that function in rice, Tos17, karma (see Patent Document 1), mPing (see Patent Document 2 and Non-Patent Document 1), and the like have already been reported.

  The transposon display method is a method for examining the function of a gene by utilizing the above-mentioned properties of the transposon, and a method for identifying a gene associated with a certain trait from the disruption of the gene caused by the transposon and the accompanying change in the trait. Specifically, this is performed as follows (see Patent Document 2).

  First, genomic DNA is extracted from a plurality of individuals of the same strain whose traits have changed due to transposon translocation, and a DNA fragment containing a part of the transposon is amplified. Next, the amplified DNA fragment is electrophoresed to obtain waveform data for each individual, and the difference in waveform data for each individual, that is, the difference in presence or absence of a peak, is analyzed. Finally, a specific trait is associated with a specific gene by comparing the difference in the presence or absence of the peak of each individual with the difference in trait.

  Here, amplification of a DNA fragment containing a part of the transposon includes (1) DNA extraction step, (2) adapter addition step, (3) first PCR step (hereinafter referred to as first PCR step), (4) It can be roughly divided into each step of the second PCR step (hereinafter referred to as the second PCR step). The details can be schematically shown in FIG. 7, for example.

(1) DNA extraction step In this step, genomic DNA is extracted from each individual of the same strain with different traits by a simple method (see Non-Patent Document 2). FIG. 7 (a) shows a genome extracted from an individual. The DNA is schematically shown. In the figure, T means a transposon, and IR means an inverted repeat.

(2) Adapter addition step In this step, the extracted genomic DNA is partially digested with a restriction enzyme, and oligomer DNA (adapter) that can bind to the recognition site by this restriction enzyme is ligated to the partially digested DNA. FIG. 7 (b) shows a state before ligation, and FIG. 7 (c) shows a state after ligation. As shown in this figure, the partial digest of genomic DNA is a mixture of partial digest 1 containing part of transposon T and partial digest 2 not containing this, and the adapter is ligated to either of them. The product is also a mixture of 3 with and without 4 part of transposon T.

(3) First PCR step As shown in FIG. 7 (c), the first PCR step consists of primer 1 specific for a part of the base sequence of transposon T and primer 2 complementary to the adapter. This is a step of performing a two-sided PCR reaction using two primers, and obtaining a PCR product as shown in FIG. 7 (d). Here, primer 1 and primer 2 are annealed to ligation product 3, and PCR product 5 is generated. On the other hand, since primer 1 cannot be annealed to ligation product 4, primer 2 is annealed at both ends, and PCR product 6 is generated.

(4) Second PCR step As shown in FIG. 7 (d), the second PCR step uses PCR products 5 and 6 as a template, a labeled primer 3 specific for a part of transposon T and the adapter. This is a step of performing PCR reaction using complementary primer 2. In addition, the labeled primer 3 and the primer 2 are annealed to the PCR product 5 including a part of the transposon, and a labeled PCR product 7 is generated. On the other hand, since the primer 3 cannot be annealed to the PCR product 6, the primer 2 is annealed at both ends to generate the PCR product 8.

  Here, as shown in FIG. 7 (e), the amplified DNA fragment is a mixture of labeled PCR product 7 and unlabeled PCR product 8. In addition, both ligation products 3 and 4 have adapters at both ends, and primer 2 which is a primer complementary to this adapter is used in both the first PCR step and the second PCR reaction. Therefore, the ratio of the labeled PCR product 7 to the unlabeled PCR product 8 in the PCR product is almost the same as the ratio of the DNA fragment 1 containing a part of transposon T and the DNA fragment 2 not containing this.

  As described above, the DNA fragment thus obtained is used for electrophoresis, analysis of waveform data, and association between traits and genes. In the past, analysis of waveform data obtained by electrophoresis has been performed manually based on the experience and intuition of the experimenter. Therefore, it requires a lot of time and labor, and the analysis results are not objective because there are individual differences. Therefore, the inventors created and used the “Rice Transposon Analysis Support System” by the “Wakayama Prefectural Regional Collaborative Research Project” in order to easily and objectively compare the waveform data obtained by different electrophoresis. (See Patent Document 3).

  In this system, size markers and PCR products are mixed and electrophoresed to convert the migration time that makes up the waveform data into the size (number of bases) of the amplified product, and to quantify the fluorescence intensity of the waveform data. be able to. Therefore, waveform data based on different electrophoresis can be corrected as if it were obtained by the same electrophoresis, and comparison of waveform data can be performed easily and objectively.

  As described above, the transposon display method is a useful method for associating a certain trait with its related gene, and its usefulness has been improved by using the above-mentioned system.

However, since the intensity of each peak constituting the waveform data is weak and the attenuation is intense, the portion of the obtained waveform data longer than 300 bp cannot be distinguished from the electrical noise peak due to electrophoresis. There was a problem that it could not be used for analysis. In addition, since the intensity of each peak is weak and the detection is unstable, there is a problem that the experimenter makes a difference in the results, and a considerable amount of skill is required before stable results can be obtained. there were.
Japanese Patent Application No. 2002-369691 US Pat. No. 6,420,117 Japanese Patent Application No. 2005-237438 Nakazaki, T., Mobilization of transposon in the rice genome, Nature, 421 (2003), p.170-172 Saya Yoshida, Kuniko Shio, “Simple rice extraction method from rice grain and varieties discrimination of sake rice by RAPD”, [online], China Agricultural Experiment Station, 1998 research result information, [May 10, 2006 search], Internet < URL: http://www.affrc.go.jp/seika/data_cgk/h10/seibutu/cgk98004.html>

  Therefore, the present invention relates to a method for amplifying a DNA fragment used in a transposon display method, wherein the peak of waveform data obtained by electrophoresis of the DNA fragment is strong and difficult to attenuate, and the longer part of the waveform pattern. It is an object of the present invention to provide a method for amplifying a DNA fragment that can be used for analysis and has little individual difference in experimental results.

  The inventor examined the reason why the intensity of the peaks constituting the waveform data is weak and the attenuation is severe. As a result, the inventor amplified not only the labeled PCR product 7 but also the unlabeled PCR product 8 at the same rate in the first PCR step and the second PCR step. It was thought that this was because nucleic acid monomers (dNTP) and the like were wasted in amplification of the unlabeled PCR product 8 and amplification of the labeled PCR product 7 was not performed corresponding to the number of cycles.

  Therefore, the inventor studied to solve the above problem by amplifying only the labeled PCR product, that is, by improving the ratio of the labeled PCR product 7 in the PCR product (hereinafter, S / N ratio). . As a result, when amplifying the DNA used for transposon display, instead of performing the normal double-sided PCR reaction using two primers twice, the first PCR reaction is performed only with primers specific to the base sequence of the transposon. It was found that the S / N ratio was greatly improved by replacing the one-side PCR method using

  That is, the DNA fragment amplification method of the present invention comprises (1) a DNA extraction step for extracting genomic DNA from cells of a higher plant, and (2) a DNA fragment obtained by partially digesting genomic DNA with a restriction enzyme. An adapter addition step of ligating an adapter capable of binding to the recognition site of the restriction enzyme, and (3) a one-sided PCR method in which the adapter is ligated and a DNA fragment is used as a template, and only a primer specific for the transposon is used. A first PCR step for amplifying a DNA fragment, and (4) both sides using a DNA fragment amplified by the first PCR step as a template and a labeled primer specific for the transposon and a primer complementary to the adapter. A second PCR step for amplifying a DNA fragment by the PCR method is included in this order.

  By the method for amplifying a DNA fragment according to the present invention, the S / N ratio of a PCR product used in the transposon display method is improved, and the intensity of peaks constituting the waveform data is strong and difficult to attenuate. Longer parts can be used for analysis, and individual differences in experimental results have been reduced. For this reason, it has become possible to more easily and objectively associate traits and related genes, thereby contributing to the growth of better higher plants.

  The method for amplifying a DNA fragment according to the present invention is similar to the conventional method for amplifying a DNA fragment, (1) DNA extraction step, (2) adapter addition step, (3) first PCR step, (4) second These steps are included in this order. Therefore, the details of each process will be described below based on FIG.

(1) DNA extraction step In this step, genomic DNA with a small number of cleavage sites, that is, a sufficiently long genomic DNA is extracted from each individual higher plant of the same line with different traits. Moreover, as an extraction method, if the said conditions are satisfy | filled, it will not specifically limit and a well-known method can be used. Specifically, a CTAB method, a simple method (see Non-Patent Document 2), and the like can be given. The extracted genomic DNA is schematically shown in FIG.

  Here, the higher plant can be used without particular limitation as long as the mutant has been obtained to such an extent that the transposon display method can be conventionally applied. Specifically, from an academic point of view, Arabidopsis thaliana and morning glory that have already undergone genetic analysis are preferred, and from an economic point of view, cereals are preferred, and among them, genetic analysis is proceeding. Rice is preferable because it has advantages such as a small genome size, a small number of repetitive sequences, and easy application of the transposon display method.

  The transposon has a known base sequence and can be used in principle as long as it transposes by natural cultivation or tissue culture to destroy the gene. Specific examples include Tos17, karma, and mPing described above. Among them, a relatively large number of copies in one individual (about 100 to several hundred copies), and an appropriate translocation activity even under natural cultivation conditions ( It is preferable to use mPing, which shows a dislocation of several to a dozen copies per generation).

(2) Addition of adapter In this step, genomic DNA is partially digested with a restriction enzyme to form a DNA fragment, and an adapter capable of binding to the restriction enzyme recognition site is ligated to this DNA fragment.

  First, genomic DNA is partially digested into DNA fragments using restriction enzymes. Here, the restriction enzyme can be used without particular limitation as long as the recognition site is present in the transposon among commercially available restriction enzymes, but it has a large number of recognition sites and is small, mainly 600 bp or less. The use of a 4-base recognition enzyme (such as Csp6I) that produces fragments is preferred. In addition, the restriction enzyme reaction may be performed according to reaction conditions such as a buffer solution and temperature required by the restriction enzyme to be used.

  Next, the partially digested DNA fragment and the adapter are mixed and ligated to the end of the DNA fragment. The adapter is a double-stranded DNA of about 10 to 20 bases having a sequence corresponding to the restriction enzyme recognition site, which is synthesized by a commercially available DNA synthesizer or the like. The ligation may be performed according to the reaction conditions required by the ligase used. Incidentally, partial digestion and ligation are not separate and may be performed simultaneously.

  FIG. 1 (b) shows a state before ligation, and FIG. 1 (c) shows a state after ligation. Here, the partial digest of genomic DNA is a mixture of one containing part of transposon T and one 2 not containing, as shown in FIG. The adapter also ligates to either. Therefore, the ligation product is a mixture of the ligation product 3 containing a part of transposon T and the ligation product 4 not containing this as shown in FIG. 1 (c).

(3) First PCR step In this step, a one-sided PCR reaction is carried out using only the primer 1 that specifically anneals to transposon T using the ligation products 3 and 4 as templates.

  As shown in FIG. 1 (c), the ligation product 3 has a nucleotide sequence that specifically anneals to transposon T. Therefore, in the first PCR step, the ligation product 3 is amplified by the number of cycles of the PCR reaction by the PCR reaction using the ligation product 3 as a template and the primer 1 as a starting point, and the PCR product 5 is generated. On the other hand, since the ligation product 4 does not have a nucleotide sequence that specifically anneals to transposon T, the primer cannot be annealed in the first PCR step, and the DNA fragment 4 is not amplified. That is, the ratio of the PCR product 5 containing transposon T is greatly improved by the first PCR step. The DNA fragment 4 and the DNA fragment 6 are the same, but are given different numbers for convenience in illustration.

  In addition, primer length, nucleotide sequence, PCR reaction conditions, specifically heat denaturation step, annealing step, extension step temperature and reaction time, number of reaction cycles, etc., depend on the target template and primer. You can adjust it while looking at the experimental results. However, as the number of reaction cycles is increased, the ratio of the PCR product 5 in the PCR product is improved.

(4) Second PCR step As shown in FIG. 1 (d), using PCR products 5 and 6 as a template, the same primer as that used in the first PCR step or specific inside thereof (transposon T side) In this step, a double-sided PCR reaction is carried out using a simple labeled primer 3 and a primer 2 complementary to the adapter.

  Since both the labeled primer 3 and the primer 2 are used in the second PCR process, the labeled primer 3 and the primer 2 are annealed in the PCR product 5 as shown in FIG. Once done, a labeled PCR product 7 is generated. On the other hand, the labeled primer 3 cannot be annealed to the DNA fragment 6 and the primer 2 is annealed at both ends, so that a non-PCR product 8 is generated. Thus, in the second PCR step, both PCR products 5 and 6 are amplified. Therefore, between the first PCR step and the second PCR step, the ratio of the DNA fragment containing a part of transposon T and the DNA fragment not containing this is almost the same.

  The labeled primer 3 is a primer labeled with a labeling substance such as a fluorescent substance, and any labeling substance and labeling method can be used without particular limitation as long as they are known. Further, the primer length, base sequence, and various conditions for the PCR reaction may be adjusted while looking at the experimental results as in the first PCR step.

  As described above, since the DNA fragment amplification method of the present invention amplifies only a DNA fragment containing a part of transposon T in the first PCR step, the ratio of labeled PCR product 7 in the PCR product (S / N Ratio) is improved, the intensity of the peaks constituting the waveform data is strong and difficult to attenuate, and the longer part of the waveform pattern can be used for analysis. Therefore, the association between the presence / absence of a DNA fragment and the difference in trait, that is, the association between a certain trait and its related gene can be performed more easily and objectively than in the conventional method.

  In addition, this invention is not necessarily limited to the said embodiment, A various change can be added within the technical scope as described in a claim. For example, other steps may be added as necessary. Specifically, a PCR reaction set consisting of a one-sided PCR reaction or a one-sided PCR reaction and a two-sided PCR reaction may be added before the PCR reaction using the labeled primer. Thus, the S / N ratio can be further improved by repeating the one-side PCR reaction a plurality of times.

  In addition, a step of inactivating / removing enzymes such as restriction enzymes, ligases, and DNA polymerases coexisting with DNA fragments and PCR products by phenol / chloroform extraction or the like, if necessary, between the above steps, ethanol precipitation A step of purifying these DNAs may be included.

  Hereinafter, although this invention is demonstrated in detail based on an Example, the claim of this invention is not restrict | limited in any meaning.

  In the transposon display method, the effect of improving the amplification efficiency of a DNA fragment containing a part of transposon T by one-sided PCR reaction was investigated. Specifically, (a) When only the first PCR step is a one-sided PCR reaction, (b) When only the second PCR step is a one-sided PCR reaction, (c) When one-sided PCR reaction is performed only once (D) Comparison with the result by the conventional method was performed.

(1) Plant material and DNA extraction
DNA extracted from 5 individual leaves of rice fine grain line IM294 planted in the field in 2004 by a simple method (see Non-Patent Document 2) was used as a sample.

(2) Adapter addition step First, an adapter was prepared by associating synthetic oligo DNAs, Csp6I-A1 and Csp6I-A2.
The base sequences of adapters such as Csp6I-A1 and Csp6I-A2 and the primer oligo DNAs described below are shown in Table 1. Next, 10 ng of genomic DNA of each individual was placed in an Eppendorf tube, partially digested with the restriction enzyme Csp6I, and an adapter addition reaction was performed using T4 ligase (Ligation high, TOYOBO). After completion of the addition reaction, Csp6I and T4 ligase were inactivated, and the reaction product was purified and concentrated by ethanol precipitation and dissolved in 40 μl of TE (pH 8.0) buffer.

(3) First PCR step A part of the reaction product (1 µl, DNA 250 pg) is collected for each individual, and the first PCR step is performed with the primer combinations shown in Table 2 (concentration is 25 pmol / µl). It was. In Table 2, Srt-P1 is an mPing-specific primer, Srt-P3-D4 is labeled with a fluorescent dye (D4, BECKMAN COULTER, USA) and is a primer specific for mPing, and Csp6I-AP Is a primer complementary to the adapter. Moreover, while showing the solution composition at the time of performing PCR reaction, Table 4 shows the PCR cycle of PCR reaction.

(4) Second PCR step The PCR product of the first PCR step is diluted to 2.5 with TE (pH 8.0) buffer, and the second PCR step is performed using the primers shown in Table 2 as a template. went. The PCR product was purified by ethanol precipitation. In addition, the solution composition and reaction cycle at the time of performing PCR reaction were the same as the 1st PCR process.

(5) Electrophoresis The PCR product obtained in this way was 0.375 μl of size marker (CEQ DNA Size Standard Kit-600 (BECKMAN COULTER, USA), labeled with D1) and sample lysis solution (CEQ Sample Loading Solution). (BECKMAN COULTER, USA) Dissolved in 30 μl and applied to a sample plate of a capillary DNA sequencer (CEQ 2000 Fragment Analysis System, BECKMAN COULTER, USA). Was converted to text data format by computer software attached to the DNA sequencer, where the waveform data is the “fluorescence intensity” at a specific “migration time”.

(6) Waveform data analysis This text data is input to the “Rice transposon analysis support system”, and each waveform data obtained from (a) to (c) by the “variation comparison” of the system and obtained by the conventional method. Comparison with the waveform data. The results are shown in (a) to (c) of FIG. In the figure, solid lines indicate waveform data from (a) to (c), and dotted lines indicate (d) waveform data according to the conventional method.

(7) Experimental results (a) When only the first PCR step is a one-sided PCR method, the maximum value of the fluorescence intensity is about 20,000, as shown in FIG. It was possible to observe a peak over a wide range. In addition, the fluorescence intensity decayed more slowly than the PCR product obtained by the conventional method, and a larger amplified product tended to be obtained. In this method, unlike the conventional method, the difference in success rate by the experimenter was small, and it was easy to learn the experimental technique. Specifically, even a beginner completed the operation within a few days, and the results were stable even after 5 iterations. In the case of the conventional method, eight people worked on, and only one person obtained a result above a certain level. On the other hand, with this method, 7 people worked on and 7 people got satisfactory results.

  (B) When only the second PCR step was the one-sided PCR method, the fluorescence intensity decreased over the entire range as shown in FIG. 2 (b), particularly at 200 bp or more. This is considered to be because primers and monomers are significantly consumed during the second PCR process as a result of the amplification of a large number of DNA fragments not containing a transposon during the first PCR step. In the first PCR step, since the ratio of the target fragment 3 and the unnecessary fragment 4 in FIG. 1 (c) does not change, the target fragment 3 has priority over the exponential amplification of the PCR product in the second PCR step. It is thought that there is nothing to do with it.

  (C) When the one-side PCR reaction was performed only once, as shown in FIG. 2 (c), the apparent attenuation was less than that of the PCR product obtained by the conventional method. One-sided PCR reactions are shown to be effective in amplifying large fragments. However, since only one type of mPing-specific primer is used in this method, there is a possibility that the target sequence may not be sufficiently narrowed down. In fact, this is probably because the number of amplified fragments in (c) is larger than the number of amplified fragments in (a).

  From the above results, it was found that (a) when only the first PCR step is unilateral PCR, a PCR product containing a part of transposon T can be obtained at a higher rate than the conventional method. Therefore, the following identification of DNA fragments containing genes related to economic traits was attempted by the transposon display method using this method.

(1) Cultivation test and DNA extraction
Individuals with non-fine grains and gold-hull traits were obtained from the ML fine grain lines cultivated in the field in 2004. Since such individuals do not occur in the breeding of fine-grained individuals and normal-grained individuals, it can be presumed that they are mutants induced by mPing transfer.

  The individual next generation lines (MLR line, all 9 lines) of these individuals were transplanted to the field. The heading date was investigated for all the plants planted, and after about one week after heading, the head length, head length, number of ears, presence of leaf hair, etc. were investigated.

  After investigating all the traits, the leaves under one of the stationary leaves were collected by hand for each individual, and genomic DNA was extracted for each individual by the simple method described in Example 1.

(2) Transposon Display and Waveform Data Analysis Using the obtained genomic DNA of each individual, the transposon display method and waveform data were analyzed in the same manner as in Example 1. Only the PCR cycle of the PCR reaction was changed to that shown in Table 5. The association between the found mutation and the mPing insertion polymorphism was then tested to detect mPing copies associated with the mutated trait.

(3) Results of cultivation test First, when the traits of all individuals were investigated in all 9 MLR lines, mutations were observed in traits such as heading time and culm length in all lines. For example, FIG. 3 to FIG. 5 show variations in heading date, culm length, head length, and number of heads of MLR4 and MLR8 belonging to the MLR line. FIG. 3 is a diagram comparing the heading date and culm length of these two lines. As shown in this figure, mutations of 30 days or more were observed on the heading date, and 30 cm or more were observed in the culm length. 4 and 5 are diagrams showing the distribution of the ear length and the number of ears, respectively. As shown in these figures, a variation of 8 to 22 cm is observed in the ear length, and the number of ears is 1 to 16. Book mutations were noted. From the above results, it was clarified that genetic variation exists in these two strains.

(4) Results of transposon display method It was possible to confirm a peak indicating amplification of the PCR product in all individuals, and also confirm a difference in peaks generated from each individual belonging to the same strain. For example, in the case of MLR8, all 79 individuals obtained, in the case of MLR4, 70 individuals excluding 8 individuals that lacked the gold-hull trait that is an indicator of illegal crossing and 4 individuals for which data was not obtained By using it, a peak indicating amplification of the PCR product was confirmed in the range from 80 bp to 600 bp in any strain, and the difference in peak between each individual was also confirmed.

  FIG. 6 is an example of a diagram comparing the differences in peaks between individuals belonging to the same strain. Specifically, the data (partial) of MLR4-2 individuals (indicated by dotted lines) as the reference individuals in the MLR4 strain and MLR4-79 individuals (hereinafter referred to as comparative individuals, indicated by solid lines) different from that were compared. It is a result. In this figure, the vertical axis indicates the fluorescence intensity (relative value) proportional to the amount of the DNA fragment, and the horizontal axis indicates the size (bp) of the DNA fragment. In this figure, the reference individual MLR4-2 has a peak and the comparison individual MLR4-79 does not have a peak, (-), and the reference individual has no peak and the comparison individual has a peak. The location where there is is indicated by (+).

  In other words, (−) indicates that mPing was inserted in the reference individual and the PCR product was amplified, or mPing was cut out in the comparative individual and the PCR product was not amplified. On the other hand, (+) indicates that mPing was cut out in the reference individual and the PCR product was not amplified, or mPing was inserted in the comparative individual and the PCR product was amplified. That is, (−) and (+) indicate mPing insertion polymorphism between the reference individual and the comparative individual.

  As described above, as a result of transposon display, the peak increase / decrease between the reference individual and the comparative individual, that is, the insertion polymorphism of mPing could be summarized in a list. Table 6 shows a part of the summary of MLR4. In addition, MLR4-2 was used as a reference individual.

(5) Integration of trait data and insertion polymorphism The trait data obtained from the field survey was compared with the insertion polymorphism of mPing, and the insertion polymorphism of mPing co-segregating with the mutant trait was examined. As a result, mPing insertion polymorphism significantly correlated with the presence or absence of mutation at 5% level for 4 traits (5a) presence or absence of leaf hair, (5b) number of spikes, (5c) spike length, and (5d) culm length. I found

(5a) Presence / absence of leaf hair DNA fragments related to the presence / absence of leaf hair were searched based on whether the presence / absence of the DNA fragment had a significant difference in the presence / absence of leaf hair. Specifically, a null hypothesis is established that there is no association between the presence or absence of leaf hair and a specific DNA fragment, the significance probability p is calculated, and if the significance probability is 5% or less Whether or not there is a relationship was determined by a χ ² test that rejects the null hypothesis (there is a relationship between the two). Table 7 shows the results that were recognized as related. The p value in this table means the significance probability p.

  As can be seen from the table, three candidate DNA fragments related to the presence or absence of leaf hair were detected with MLR4 and three with MLR8. Among these, a 120 bp fragment could be detected in both lines, and the p value was as low as almost 0%. From this, it can be considered that hairlessness is expressed by excising mPing from this locus.

(5b) Number of spikes Specific DNA fragments related to the number of spikes were searched for by t-test based on whether the presence or absence of the DNA fragment gives a significant difference in the number of spikes. Specifically, a null hypothesis is first established that there is no association between the number of spikes and a specific DNA fragment. The variances were calculated respectively, and the index t was calculated based on them. Next, the significance probability p was obtained from the index t and the degree of freedom, and when the significance probability was 5% or less, the null hypothesis was rejected (there is a relationship between the two). As a result, as shown in Table 8, 4 DNA fragment candidates and 3 MLR8 DNA fragment candidates related to the presence or absence of the number of spikes were detected. The p value in this table means the significance probability p.

(5c) Ear length Regarding the head length, a specific DNA fragment related to the head length was searched for by t-test as in (5b). As a result, as shown in Table 9, 6 DNA fragment candidates and 3 MLR8 DNA fragment candidates related to the ear length mutation were detected.

(5d) Saddle length For the heel length, similar DNA fragments were searched for by t-test as in (5b). As a result, as shown in Table 10, five candidate DNA fragments and four MLR8 DNA fragment candidates related to the ear length mutation were detected.

  Thus, since the S / N ratio of the PCR product is improved by the DNA amplification method of the present invention, a wider range of waveform patterns can be used for analysis. In addition, since the variation of the experiment results by the experimenter has decreased, the association between the trait and the gene by the transposon display method can be performed more easily and objectively.

BRIEF DESCRIPTION OF THE DRAWINGS It is the schematic which shows the outline | summary of the amplification method of the DNA fragment concerning this invention. It is the figure which compared the change of the waveform data by the difference in the structure of PCR method. It is the figure which compared the heading time and culm length of MLR4 and MLR8 system | strain. It is a figure which shows distribution of the panicle length of MLR4 and MLR8 system | strain. It is a figure which shows distribution of the number of spikes of MLR4 and MLR8 system | strain. It is an example of the figure which compared the difference in the peak of the individuals which belong to the same system | strain. It is the schematic which shows the outline | summary of the conventional amplification method of a DNA fragment.

Claims (3)

A method for amplifying a DNA fragment used in a transposon display method for higher plants,
(1) a DNA extraction process for extracting genomic DNA from cells of higher plants;
(2) an adapter addition step of partially digesting genomic DNA with a restriction enzyme to form a DNA fragment, and ligating an adapter capable of binding to the restriction enzyme recognition site to the DNA fragment;
(3) a first PCR step of amplifying the DNA fragment by a one-side PCR method using the DNA fragment to which the adapter is ligated as a template and using only a primer specific for the transposon;
(4) Second DNA amplification by a double-sided PCR method using the DNA fragment amplified in the first PCR step as a template and a labeled primer specific for the transposon and a primer complementary to the adapter PCR process;
A method for amplifying a DNA fragment comprising   The method for amplifying a DNA fragment according to claim 1, wherein the higher plant is rice.   The method for amplifying a DNA fragment according to claim 1, wherein the transposon is mPing.

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