WO2001022074A1

WO2001022074A1 - Highly efficient method of genome scanning

Info

Publication number: WO2001022074A1
Application number: PCT/JP2000/006512
Authority: WO
Inventors: Shinji Kawasaki; Takao Komatsuda; Yoshiro Mano
Original assignee: National Institute Of Agrobiological Sciences
Priority date: 1999-09-24
Filing date: 2000-09-22
Publication date: 2001-03-29
Also published as: CN1399721A; CA2385943A1; JP3573130B2

Abstract

It is found out that use of a nucleic acid electrophoresis apparatus with the use of a small-sized gel makes it possible to detect polymorphic bands at a remarkably higher efficiency than in the conventional methods. It is further found out that this method is appropriately usable in analyzing heredity, distinguishing biological strains, etc.

Description

Description High efficiency genome scanning

The present invention relates to a method for electrophoresing a large number of nucleic acid samples with high efficiency to detect a target nucleic acid, and an electrophoresis apparatus used in the method. The method of the present invention is particularly useful in the detection of polymorphisms in genomic DNA, genetic analysis, genetic mapping, and the creation of contigs or physical maps covering the entire genome of an organism. Background art

Until now, when trying to detect differences between biological varieties at the nucleic acid level, techniques such as the R FLP (Restriction Fragment Length Polymorphism) method and the EAPD (Randomly Amplified d Polymorphic DNAs) method have been mainly used. Was. However, the RFLP method requires a large amount of DNA samples, and a genome map based on existing RFLP markers for the organism is indispensable for searching for markers near a specific gene. . Moreover, the construction of this map requires a long time, enormous cost and manpower, and very few organisms have genetic maps with sufficient density and RFLP markers. On the other hand, polymorphism detection by the RAPD method is easily applicable to a relatively large number of samples, but the number of stable bands obtained at one time is limited to only a few. In this case, if the number of bands obtained is increased by relaxing the PCR conditions, there is also a problem that the reproducibility of the obtained polymorphic band is reduced.

Recently, the Amplified Fragment Length Polymorphism (AFLP) method can be used at a time because many (50-100 or more) bands can be compared at once, the DNA consumption of the sample is small, and the reproducibility of the obtained band is high. It has become. However, this original method uses a large sequence gel of 40-50 cm, This is a task that requires much experience, such as detection of nucleic acid bands by tradography, and also has limited use conditions, takes a long time to detect, and cannot analyze a large number of samples (one time). Tens).

Recently, a relatively simple method of detecting bands by PCR using fluorescent primers and an automatic sequencer has been developed. However, the sequencer used in this method is expensive (more than 1-2 million yen). In addition, experiments using this method take a long time, and occupy the machines that are used to determine the nucleotide sequence of genes. Furthermore, since band detection in this method is premised on being performed in a system using an automatic sequencer, the band cannot be isolated and analyzed after detection. For this reason, a major problem is that the detected band cannot be easily detected with a specific primer as an SCAR (Sequence Characterized Amplified Region) marker. In addition, currently available fluorescent primers are 8-9 types, and even if they are all combined, only a combination of 64-81 primer pairs can be obtained.

Another method for detecting polymorphic bands in the whole genome at one time is the RLGS (Restraction Landmark Genome Scanning) method, which also uses radioisotopes to detect trace markers. In addition to this, it takes several days, and it is necessary to handle a huge (40 x 30 cm) gel or a long and thin agarose gel per sample. For this reason, this method requires strength and sufficient experience. In addition, this method requires the use of a large amount of expensive restriction enzymes in order to cleave the nucleic acids in an agarose gel, which involves a high cost.

For example, when the RLGS method is performed on a rice genome (450 MB), only a limited number of 8-base cleavage enzymes, such as Not I, can be used to limit the labeling site. a dot upper theoretical value ^{450 MB ÷ 4 8 = 13,} 700 of Bok obtained by electrophoresis in fact of the nature of the electrophoresis, dot of the 1 / 3-1 / 6 of about 2,000 to 4,000 I can't get it. Moreover, since the individuals to be compared are electrophoresed on different gels, In many cases, migration patterns are displaced, and an expensive large-sized scanner and two-dimensional migration software are required for mutual comparison.

On the other hand, in the early days of creating a genomic library in which adjacent clones covering the entire genome were arranged and connected by overlapping each other, attempts were made to use markers on existing maps such as RFLP maps. However, even a high-density map has at most about 2,000 markers, and a small genome (450 MB) as small as rice has an average density of 200 KB / band or more. However, it is too sparse to create a contig covering the entire genome, and searching for this many RF LP markers requires enormous manpower, cost and time.

Recently, fragments obtained by cleaving library-constituting clones with appropriate restriction enzymes are electrophoresed on a high-resolution gel, all the patterns are digitized and imported as a database. A method of assembling a contig covering all genomics is often used. However, even in this method, enormous labor and cost of performing autoradiography by cleaving all clones and radiolabeling the end thereof are required. Disclosure of the invention

The present invention has been made in view of such circumstances, and a purpose thereof is to efficiently and inexpensively electrophorese a large amount of a nucleic acid sample, to detect a target nucleic acid, and to provide a method for the method. To provide an electrophoresis apparatus. Further, the present invention provides, in a preferred embodiment of the method, a method for detecting a genomic DNA polymorphism using the method, a method for genetic analysis, a method for preparing a genetic map, a method for identifying a genomic clone for a specific band, and a method for identifying a whole genome. A method of making an ordered collection of contigs covering a system is provided.

The present inventors have conducted intensive studies in order to solve the above problems, and as a result, an electrophoresis method using a small gel conventionally used for protein electrophoresis has been proposed. We thought that a large amount of nucleic acid could be efficiently electrophoresed and used suitably to detect the target nucleic acid.

Thus, the present inventors have developed an electrophoresis apparatus for electrophoresing nucleic acids, which can be equipped with a plurality of 10-30 cm square gel plates at the same time to perform electrophoresis. An electrophoresis device capable of simultaneously electrophoresing 32 or more nucleic acid samples was manufactured independently. Using this electrophoresis device, the non-pathogenic gene of the rice blast fungus and the rice camillaz mutant gene were A search for nucleic acid markers was performed. As a result, they found that this method can detect polymorphic bands with remarkably high efficiency as compared with the case of searching by the conventional RAPD method.

In addition, as a result of performing a linkage analysis on the detected polymorphic bands, among the detected polymorphic bands, in particular, those located near the target gene, this method is far more effective than the conventional method. Was found to be efficiently obtained.

In addition, this method was shown to be useful in isolating and specifically amplifying various important polymorphic bands found by the above-mentioned method. As an example, the present inventors have isolated a large number of bands identifying 10 varieties for rice milling by this method, and in fact, designed a primer from sequence information of a band specific to Akitakomachi therein, Genomic DNA obtained from 10 rice cultivars was subjected to PCR using type II. As a result, they found that a specific amplification product by PCR was detected only in Aki Takomachi. That is, it was shown that according to the method of the present invention, a polymorphic band for easily distinguishing rice varieties can be efficiently obtained.

Furthermore, the present inventors have found that this method can also be used to prepare a genetic map of an organism and obtain a nucleic acid matrix for a contig covering the entire genome of an organism.

Therefore, the present invention relates to a method for efficiently and inexpensively electrophoresing and detecting a large amount of a nucleic acid sample, an electrophoresis apparatus for the method, and use thereof, and more specifically, (1) A method for electrophoresing nucleic acids,

(a) An electrophoresis apparatus capable of simultaneously carrying out electrophoresis with a plurality of gel plates of 10 to 30 cm square, and capable of simultaneously carrying out electrophoresis of 32 or more nucleic acid samples per gel plate Electrophoresing a nucleic acid sample using

(b) detecting a nucleic acid band on the gel after electrophoresis, a method comprising:

(2) performing the electrophoresis using a discontinuous buffer type gel, the method according to (1),

(3) Single-stranded nucleic acid sample prepared by dissociation of double-stranded DNA by denaturation treatment! The method according to (1), wherein the electrophoresis is performed using a denaturing gel,

(4) Detecting the nucleic acid node on the gel by fluorescent staining or silver staining.

The method described in (1),

(5) To detect genomic DNA polymorphisms among test subjects, (1),

The method according to any one of (2) and (4),

(6) The method described in (3), which is performed to detect a genomic MA polymorphism between test subjects,

(7) The method according to (5), wherein the nucleic acid sample is a DNA fragment amplified by the AFLP method.

(8) The method according to (5), wherein the nucleic acid sample is hetero double-stranded DNA.

(9) A method for preparing a DNA fragment exhibiting a polymorphism, comprising isolating, from a gel, the DNA fragment exhibiting the polymorphism detected by the method according to any one of (5) to (8). Including the process,

(10) A DNA fragment showing polymorphism among test individuals isolated by the method described in (9),

(1 1) The method described in any one of (1) to (8), which is performed to perform genetic analysis,

(12) The method according to (11), wherein the genetic analysis is F2 analysis, RI (recombinant imbred) analysis, or QTL (Quantitative Traits Loci) analysis. (13) The method according to any one of (1) to (8), which is performed for preparing a genetic map of an organism,

(14) A genetic map of an organism created using a genomic DNA band showing a polymorphism detected by the method according to (13) as a marker,

(15) A method for selecting a clone corresponding to a specific nucleic acid band on a gel detected by the method according to any one of (1) to (8) from a genomic DNA library,

(a) dividing a genomic DNA library of a particular organism into a plurality of sub-libraries, each of which is smaller than one genome of the organism;

(b) The process of assigning the number of the row, column, and plate of the supply library to all the clones included in each subbrief (here, the row, column, and plate are designated as coordinate X, coordinate Y, and coordinate Ζ, respectively). Do),

(c) A clone indicating a particular row of all plates (coordinates X clones), and a clone indicating a particular column of all plates (coordinates Υ clones), and on a particular plate of one supply library All the clones (coordinates Ζ clone group) are collected separately, DN 抽出 is separately extracted and used as a coordinate sample, genomic DNA prepared from the organism is prepared as a control, and the coordinate sample and the control are arranged side by side. Electrophoresis by the method described in any one of 1 to 4, and detecting a band,

(d) Bands with the same mobility on the gel as the band of the nucleic acid of interest in the control are the clones at coordinates X, Y, and Z clones. The process of determining which coordinates are clones,

(e) selecting a clone corresponding to the determined three-dimensional coordinates from one of the sub-libraries;

(16) The method according to (15), which is performed to create a contig covering the whole genomic DNA of a specific organism. (17) An electrophoresis apparatus for electrophoresing nucleic acids, in which a plurality of gel plates of 10 to 30 cm square can be equipped simultaneously to perform electrophoresis, and one gel plate can be used. An electrophoresis apparatus capable of simultaneously electrophoresing the above nucleic acid samples.

1. Electrophoresis and electrophoresis equipment

The electrophoresis apparatus according to the present invention is an electrophoresis apparatus capable of mounting a small polyacrylamide gel (10 to 30 cm square, standard 18 cm square), and each gel contains 32 or more (64 standard) samples. Several (2-4 standard) size electrophores can be electrophoresed at the same time, and two or more (4 standard) size electrophores can be electrophoresed to obtain a large number of (256 standard) samples. Can be electrophoresed simultaneously. One example of the electrophoresis apparatus used in the present invention is shown in FIGS. 1 to 4. In this electrophoresis apparatus, a single gel made of one piece of 18 cm square glass has a thickness of 66 and has 64 samples. Two size standards can be run at the same time, and four gels can be electrophoresed simultaneously with a single swimming device. As a result, 256 samples can be simultaneously electrophoresed at one time.

For the gel, a discontinuous polyacrylamide electrophoresis system (Lae Verili, UK (1970) Nature 227: 680-685), which is usually used for protein electrophoresis, can be used. A wide range of samples can be run with high resolution even in lanes with a narrow lmm width, and the sensitivity of band detection is also improved.

In the nucleic acid electrophoresis method of the present invention, in order to improve the resolution of the nucleic acid band on the gel, an agarose gel (Tris-HC1 pH6.8, 0.5M) and a separation gel (Tris-HCl pH8) are used. .8, 1.5M) to perform two-layer electrophoresis. Regarding the electrophoresis of the nucleic acid, it is possible to migrate the nucleic acid as a double strand, or to migrate the nucleic acid as a single strand after denaturation according to the purpose. In this case, perform electrophoresis on a denaturing gel (gel containing 6 to 8.5 M urea). When detecting nucleic acid polymorphisms, By electrophoresis as a heavy chain, small polymorphisms that cannot normally be detected can be detected with high sensitivity.

In the nucleic acid electrophoresis method of the present invention, it is preferable to use a silver staining method or a fluorescent staining method for detecting a nucleic acid band after electrophoresis. Using silver staining, highly sensitive detection can be performed in a short time (1-2 hours). Moreover, compared to the method using an isotope, less experience is required, and if it is safe and dried as it is, it can be stored as a sample and the sensitivity can be further increased. If fluorescent staining is used, the results can be seen in about 30 minutes of staining, and the nucleic acid recovery rate is high and excellent for excision and recovery of bands.

In the method of the present invention, for example, an amplified DNA is designed by designing an electrophoresis gel comb so that two or more lanes can flow at the same interval (9 references) as the holes of an 8 × 12 96-well microplate. Operation efficiency when loading a sample or the like onto a gel can be greatly improved.

2. Detection of nucleic acid markers

When the polymorphism of nucleic acid is to be detected by the method of the present invention, the combination with AFLP (Amplifie d Fragment Length Polymorphism: Vos P, et al. (1995) Nucleic Acids Research 23: 4407-4414) is most preferable. Although it is considered to be highly efficient, it can be combined with other various nucleic acid polymorphism detection methods.

For example, if the AFLP method is used, a primer that amplifies a genomic fragment having a 6-base cleavage side and a 4-base cleavage side on both sides of a rice (450 MB) size genome will have 3 bases on each side. When the selected nucleotides are used, about 50 amplified bands are obtained per lane (see the following formula). In a standard 4-gel 256-sample lane system, about 12,800 bands can be obtained in one run (Example 2). Whole genome size 数 Number of cuts by 6 base restriction enzyme 選択 Selectivity by 3 nucleotides on both sides of genome fragment = Number of bands

(450MB ÷ 4 ⁶ x 2 ÷ 4 ^{3 x 2} = 50)

This is comparable to the amount of information for several gels when performing the RLGS method, and since the lanes to be compared can be arranged next to each other, it can be used in raw data without using a special reading device. You can easily compare directly.

As shown in Fig. 6 (Example 2), a band close to the number estimated by the above calculation was obtained from the actual AFLP experimental example obtained by cutting rice genomic DNA with EcoM and Msel. Although the number is obtained, about half of the bands are particularly clear.

Performing PCR at 5-10〃1 using 0.2 ml microplates for 96 samples can reduce the cost of enzymes and nucleotides such as thermostable DNA polymerase, Eight samples can be transferred to the gel at one time per bit, so efficiency is high, and electrophoresis of four gels can be performed once a day with sufficient margin.

Four gels after electrophoresis can be stained efficiently and at low cost by performing silver staining in one container at a time. Generally, a commercially available silver staining kit for proteins can be used for staining.

As a nucleotide primer used in the AFLP method, a nucleotide suitable for any restriction enzyme can be used, and an optional selected sequence of one to several bases can be added to the 3 ′ side. Almost infinite combinations are possible. For plants with a genome size of 1 GB or less, using EcoRI and Msel as the restriction enzymes of 6 bases and 4 bases, respectively, and using PCR primers having 64 3 nucleotide selection sequences each, 64 x 64 = 4,096 amplifications are possible, and in genetic analysis between parents with a polymorphism rate of 5 to 10%, 10,000 to 20,000 polymorphic bands can be searched. This is a practically sufficient number for physical mapping, and exceeds the number of markers in conventional genetic maps by more than one order of magnitude. Important bands such as those near the target gene obtained by bulk analysis (Michelmore RW, et al. (1991) Pro Natl. Acad. Sci. USA 88: 9828-9832) were cut out after staining. After the gel is crushed, it is extracted with an appropriate buffer, re-PCRed, inserted into an appropriate plasmid, ligated, and isolated to analyze its sequence (Example 4).

With the above combination, for example, a set of 4 samples consisting of 2 samples of mixed parents and 10 samples of each of dominant and recessive homozygous individuals in F2, one set of 4 lanes / primer pair If electrophoresis is performed, a standard electrophoresis apparatus (4 gels: 256 lanes) can run 64 primer pairs in one run. As a result, all combinations of the 4,096 selection primers are completed in 64 runs (about 2 months in total). This is equivalent to scanning and retrieving 20,000 polymorphic bands in the whole genome for a combination with about 10% polymorphism, such as the cross between rice and India. This efficiency is one to two orders of magnitude higher than conventional methods, which allows for the isolation of genes in a short time, with or without a genetic map. In addition, the cost to search all 4,096 entries is inexpensive, at around 400,000 yen.

3. Genetic analysis

1) F2 analysis

When trying to determine the genetic distance (or distance on the map) between any combination of genes or polymorphic markers, the most commonly used is F2 analysis, but the method of the present invention is used. According to the F2 analysis described above, the distance between polymorphic markers or between a polymorphic marker and a gene can be determined extremely efficiently.

In other words, as in general F2 analysis, a line (A) that has a certain gene and a line that does not have the target gene or has a clear difference in its traits. Select and cross with a certain line (B) to obtain a large number of F2. The number of F2 individuals to be analyzed depends on the accuracy of the analysis. If 50 individuals (usually recessive homozygotes) are used, determine the genetic distance between the target gene and the polymorphic marker by examining the recombination rate for 100 chromosomes be able to. Analysis of the recessive homozygous individuals shows that if there is no recombination, all individuals with the same marker type as the parent with the recessive trait in one parent have a recombination rate of 1/100. Will be seen.

In the present invention, since 256 individuals can be analyzed at a time using a standard system, if only such homologous individuals are obtained, it is possible to rearrange the polymorphisms on the 512 chromosome by one electrophoresis. Can be tested, and analysis with an accuracy of 0.2CM is possible. By using the AFLP method as a polymorphic marker, the test can be performed with a very small amount of sample DNA (less than 100ng). In other words, genomic DNA is prepared from each individual, and double digested with two enzymes, EcoRI and Msel, and adabu Yuichi adapted to each enzyme is ligated to the genomic DNA fragment. Perform primary PCR using a primary primer that is compatible with this adapter to amplify genomic fragments. Next, using a secondary primer in which an arbitrary 1-4 bases are extended to the 3 'side of the primary primer, only a part of the genomic fragment conforming to this sequence is subjected to PCR amplification, and the electrophoresis method of the present invention is used. Separate according to molecular weight. After the electrophoresis, stain the gel with silver stain, etc., and test whether the target polymorphic marker has been recombined.

According to the present invention, precise F2 analysis at the 0.2CM level for 256 individuals can be completed with a single run using a small amount of DNA, and no staining or autoradiography is required. Since the gel can be stored in the album as it is in a cabinet size, the analysis of the results is extremely easy.

2) RI analysis

RI strains (Recombinant Inbred lines) are strains obtained by crossing generations of different F2 individuals by crossing them for several generations. As a result of the large number of selfing, most of the loci are homozygous and the number of heterozygotes is small, so the segregation ratio is 1: 1 for the dominant trait: recessive trait. Since they are homozygous, genetic analysis according to the present invention is possible as it is. A more precise genetic analysis is possible than F2 analysis, in which the proportion of heterozygous individuals is halved and the separation ratio of dominant and recessive traits is 3: 1. If an appropriate RI strain is available, genetic analysis (RI analysis) using genomic DNA extracted from many RI strains using the present invention can be performed in exactly the same way as in the case of F2 analysis.

3) Narrowing down nearby markers

By using the electrophoresis method of the present invention, the standard method can be used to narrow down the markers near the target gene obtained by the Nork analysis method by F2 analysis and to finally map the group of the nearest markers. Since 256 samples can be run at one time, genetic analysis can be performed with extremely high efficiency. In other words, in rice (in the case of the cross between India and Japan), as described in Section 2, if the polymorphisms in the 20,000 band were evenly distributed in the whole genome of 2,000 CM, the bulk It is expected that the number of markers present in a total of 20CM, including about 10CM on both sides near the gene obtained by the method, will be 200 bands. In order to narrow down the nearest markers among these neighboring markers, as a first step, first, 14 recessive homozygous individuals of the F2 generation per marker and the DNA of their parents are passed to 16 lanes per marker. Then, the presence or absence of recombination between the target gene and the candidate marker is examined. Thus, the recombination rate for 28 chromosomes is calculated for each marker. The resolution at this time is about 3.5CM. As a result, a test of candidate markers of 4 bands per gel is performed, and it is possible to check markers of 16 bands in one run, and at the same time, recombination occurs in the nearest vicinity of the gene. Individuals that are present become apparent. After that, only 8 lanes are used in total, including 6 transgenic individuals and parents in the vicinity, and the check of the remaining 192 markers is completed by 6 runs.

Assuming that the marker and the chromosome recombination position occur evenly, it is expected that about 40 bands of markers near 8CM in total, within about 4CM on each side of the gene, can be obtained by the above screening. In the second step, the remaining markers from the primary screening are mapped to 1CM accuracy. In each run, 12 gels are used for each of the 12 markers, and the AFLP products of the 62 recessive homozygous F2 generations + parents are run on one gel. As a result, a precise map of the region within 8CM of the gene can be obtained, and individuals that have been recombined in the nearest neighborhood of less than 1CM of the gene can be identified. Analyzing the remaining 28 bands using the rearranged individuals in these neighborhoods will clarify their location on this map. Again, the analysis in the vicinity of the gene will be completed by checking the combination of 6 nearest neighbors and 8 parents x 28 combinations = 224 lanes together with parents in one run.

That is, out of 200 neighboring marker candidates selected by the bulk method from the 20,000 polymorphic bands, the nearest marker within about 1 CM can be narrowed down by about 11 runs.

In gene isolation, if an organism with a genome size of 1 GB or less has such a marker, the average 1 CM will be 500 kB or less. Therefore, from a genomic library using a BAC (Pacteria artificial chromosome) with an average insert size of about 150 kB. Contigs can be formed by selecting clones near the gene.

Even when the accuracy is further improved and analysis is performed at the 0.2 CM level using 256 recessive homozygotes, if the appropriate nearby marker is selected for about 4 bands and run four times, which individual It becomes clear whether recombination is occurring nearby. Using the recombined individuals in these neighborhoods and checking the recombination in the band considered to be the nearest in the same way as in the second stage, the nearest marker can be easily obtained by one run. Can be

Here, the principle is premised on the assumption that bands obtained by electrophoresis are distributed almost uniformly in the genome and chromosome, and that chromosome recombination occurs uniformly on the genome. In fact, the distribution of bands and recombination positions in the chromosome is unevenly distributed, so the distance between the target marker and the neighboring marker obtained for the number of F2 used is uniform. Tends to be larger than at the time. 4) Application to breeding In addition, the near marker for the specific trait gene obtained as described above is used as a highly reliable marker even when performing native breeding by crossing, and in a large number of progeny obtained by crossing. When analyzing markers, analysis using the genome scanning method can easily analyze a large number of individuals by taking only a small amount of DNA sample, which is a significant task compared to the conventional method using the RFLP method. This can improve efficiency and reduce costs.

5) Application to QTL (Quantitative Traits Loci) analysis

A QTL is a genetic trait locus in which the expression of the trait is not qualitatively strong and, in many cases, multiple loci are involved in the expression of that trait. QTL analysis analyzes which loci at which positions on the chromosome contribute to the expression of the trait.

In QTL analysis, polymorphisms in a large number of mating progeny individuals must be examined for a large number of nucleic acids that are uniformly distributed throughout the entire genome. For example, if QTL analysis is performed using markers distributed in the entire genome of 2,000 CM at a marker density of about 10 CM / line, it is necessary to examine at least about 50 individuals with 200 markers per F2. If this is performed using the RFLP marker, it is necessary to perform 200 hybridizations on a membrane on which 50 nucleic acids have been plotted, and one hybridization requires two days. Even if about 4 copies are performed at the same time, a total of 100 days will be required. Even if a membrane can be used ten times, the effort required to collect as many as 100 µg of nucleic acid from all 50 individuals to prepare 20 membranes is enormous.

By using the AFLP method in the method of the present invention, several polymorphism bands can be obtained per lane in those showing about 10% polymorphism such as rice-Japanese cross, so that an appropriate primer pair The search for 200 markers can be completed by electrophoresis with a little over 50 primer pairs. Since it is possible to run 5 primer pairs in one run (50 x 5 = 250 lanes), migration of 50 individuals After 10 or 10 runs (total of 10 days), all markers can be examined. In addition, 1 g of DNA is required for each individual.

Specifically, first, it is necessary to have a genome map of the organism with the AFLP marker, but if there is no existing map, it can be easily applied using the present invention as described in Section 5 below. Can be created. From this map, select markers at the desired density, but it is more efficient to select a marker that covers the entire genome with as few primer pairs at that time.

Regarding crossover, appropriate polymorphism frequencies can be obtained by selecting combinations that are genetically separated from each other in lines that have a remarkable quantitative trait and those that have almost no morphology. If the combination is too far apart, marker separation may not be performed smoothly. For about 50 F2 or RI strains (the number of which varies depending on the purpose) obtained from this cross, the target trait was quantitatively measured, and the genomic MA prepared therefrom was used for 3.1. As described in (1), genomic fragments are amplified by the AFLP method, and the resulting DNA is electrophoresed by the system of the present invention to check and record the polymorphism of the marker band.

By plotting the product of the number of target traits and the number of polymorphisms of one parent for each band for all marker bands on the map, the contribution to the target trait by loci near each marker Becomes clear.

4. Application to varieties and strains

According to the present invention, it is possible to efficiently search for a marker necessary for discriminating a specific breed or line of an agricultural or livestock product or various biological species. That is, AFLP is performed using DNAs obtained from a large number of varieties to be compared, and by performing electrophoresis simultaneously using the electrophoresis apparatus of the present invention, tens of bands are obtained for tens to hundreds or more varieties. Can be compared at the same time. This makes it possible to easily select a specific identification for a large number of varieties. In this case, by combining n bands, it is theoretically possible to identify up to 2n varieties and strains, but in actual cases the number of varieties and strains that can be identified is less than this Somewhat less.

When varieties and lines are very closely related and it is difficult to obtain polymorphism with normal AFLP, etc., AFLP or RAPD products from the genome to be compared are mixed, heated and cooled, and migrated as heteroduplex. By comparison with the target band, differences between varieties can be detected with high sensitivity, easily and efficiently.

If a specific band with high discriminating ability is identified, the band can be isolated by the method described in Section 7 and PCR-amplified from specific primers.After PCR, simple agarose electrophoresis can be performed for 1 hour. The type can be determined by the degree. Furthermore, a specific fluorescent label is pre-applied to the primer, and whether or not a band is amplified therefrom is checked with a PCR device equipped with a fluorescence detector. It is also possible to identify varieties and strains without performing.

5. Genetic mapping of organisms

When creating a genetic map that covers the whole genome of an organism (more precisely, a map of nucleic acid markers), the required map resolution for hundreds to over a thousand markers is the same as when performing QTL analysis. It is necessary to analyze using the number of F2 individuals corresponding to In the case of higher plants, especially the major crops, most of which have a total genome length of 1,500 to 2,000 CM, if a genome map is to be produced by the conventional RFLP method with a density and accuracy of about 1 CM, more than 100 individuals It is necessary to repeatedly plot the membranes produced from the F2 generation for about 1,800 markers, and it takes four days including the preparation of the probe in one plot. It takes 1,800 days of work to process any kind of probe. In addition, the amount of nucleic acid required for this is enormous, and each individual F2 requires lmg or more of DNA. For this reason, genetic maps have been created by teams of tens of people over many years. In the present invention, if a combination of mating parents having a polymorphism of about 10% is taken as an example, an average of 4 to 5 polymorphisms can be found in one combination per primer. A primer combination showing a polymorphism is selected, and using this, muting is performed using the F2 generation of 128 individuals in a standard model, so that two primers and a pair of 12 or more markers can be mapped in one run. Becomes possible. Therefore, a map with 1,800 markers can be made with a single person and a total of 150 days of analysis to produce a precise map, which is 10 times more efficient than the conventional method. A map with only 300 markers can be made alone in about a month.

As a specific procedure, two lines separated by an appropriate genetic distance are bred to produce F2 individuals or RI lines of the number corresponding to the accuracy of the target map. If the average polymorphism rate does not exceed 10% between both lines, there is little risk of hybrid sterility and distortion of the isolation ratio. In the meantime, if accuracy of about 0.5 or 1 CM is aimed at, sufficient accuracy can be obtained if there are 128 or 64 individuals. Genomic DNA is prepared from these individuals, and PCR fragments amplified by the AFLP method are electrophoresed, stained, and analyzed by the system of the present invention in the same manner as in the F2 analysis and RI analysis described above. That is, the prepared genomic DNA is stored in a 96-well microplate, a part (about 0.1 ug) is taken, double digested with EcoRI and MseI, and the adapter is joined to the cut surface. Perform preamplification with the same 5 'primer as the adapter. Next, the pre-amplified PCR product is subjected to secondary amplification using a selection primer having an appropriate number of selection bases as type III, and then applied to the electrophoresis apparatus of the present invention. At this time, using 8 or 12 pipes or 8-12 micro syringes will improve the efficiency of gel loading. For 64 samples, 4 primer sets can be used for about 20 markers, and for 128 samples, 2 primer sets can be used to run 10 markers at a time. For analysis of polymorphic bands, it is efficient to use genetic mapping software such as MAPL or MAPMAKER. As a reference case, using the predecessor system of the present invention using 8 gels of 15 lanes, an AFLP map of 227 markers was created in about 2 months using Azumamugi of oats and RI of Kanto Nakaseo Gold, 99 lines. Then, an example is shown in which a map consisting of 272 markers was created by integrating it with a map consisting of 45 markers obtained so far (Fig. 11). Even in this case, an AFLP map can be created alone in about two months, and about 40 times as much efficiency has been achieved as compared to the previous case of creating a map using an STS marker or the like. In the method of the present invention, the working efficiency is further improved compared to the system of the predecessor.

6-Application to species with large genome size

The present invention appears to be most effective when used in combination with AFLP, but the characteristics of AFLP are the genomic fragment digested with EcoRI (6 bases recognized) and Mse I (4 bases recognized). The adapter was connected to the fragment and adapted to the respective cutting site.In the first amplification, this adapter sequence was used as a PCR primer to amplify all fragments, and as the second primer for the next secondary amplification The point is that using a selection primer in which the selection sequence of one to several bases is extended on the third side of the primary primer, only the genomic fragments having a sequence that is compatible with the selection primer are specifically amplified. In the case of organisms with a relatively small genome size (rice: 450 MB), primers with 3 bases each on both sides of EcoRI and Msel sites are used as secondary primers. As a result, on average, about 50 bands are selectively amplified as described in “2. Detection of nucleic acid marker”. For genomes of the size of filamentous fungi such as blast fungus (40 MB), by adding only an arbitrary sequence of only one base at either restriction site, 50 lines X (40 MB / 450 MB) x 16 = 70 lines It is expected that a band will be obtained. On the other hand, in organisms having a large genome size, it is basically possible to cope by increasing the number of bases of the selected sequence in the AFLP primer from 3 to 4.

However, as another method, the electrophoresis conditions can be changed. Normally, when PCR amplification products are electrophoresed, these materials are left as double strands It is convenient to run under non-denaturing conditions, but for organisms with a larger genome size (eg, wheat: 5.5 GB), the bands are not clearly separated if the sample is run as a double strand. Here, using a denaturing gel containing 8.5 M urea, the DNA sample is subjected to thermal denaturation at 90 ° C for 3 minutes in the presence of 50% formamide to dissociate it into single-stranded DNA. Many bands can be clearly identified (Example 4). In actual situations, AFLP becomes more difficult as the genome size increases, so it is realistic to use a combination of the above two methods.

7. Isolation of important bands and their conversion to SCM markers by sequence analysis

In the implementation method of the present invention shown in 3, 4, 5, etc., a band that carries particularly important information is identified in each case. That is, a band in the vicinity of the specific gene in 3), a band suitable for cultivar discrimination in 4), and a band that becomes a landmark in the genetic map in 5).

These bands are cut out from the gel, eluted by crushing extraction, freeze-thawing, electrophoresis, etc., and then PCR-amplified again with the same primer pair, inserted into an appropriate cloning vector, sequenced, and both ends are primed. It can be a SCAE (Sequence C haracterized Amplified Region) marker (Example 3). In this case, it is easier to extract nucleic acids by using fluorescent staining such as cyber green for staining. Compared to the AFLP method using a sequencer, the method of the present invention is simple but allows full use of the essential functions of the original AFLP, as compared to the AFLP method using a sequencer. Is excellent.

8. Isolation of genomic clones containing specific bands

Bands that carry important information, such as those described in 3., 4., 5., etc., may require isolation of genomic clones containing them. In particular, when isolating a gene having an important function by positional cloning, identify the nearest neighbors on both sides of the gene using the methods described in 2. and 3. and then use them. To select a clone with a band from a genomic library such as a BAC library, The next successive clones are identified using the Kerr (walking) to create a contig of a series of clones that connect between the flanking markers on both sides of the gene. There is a need.

If a high-density membrane has already been prepared by arranging and binding the constituent clones of the genomic library by colony hybridization, as a band directly amplified by PCR as in step 7, or as a SCAR marker The target clone can be picked up by performing hybridization on the membrane using the amplified band as a probe.

Alternatively, after dividing the genomic library of the organism into subgenomic libraries by the method described in Section 9, clone the clones in the subgenomic library so as to correspond to the row, column, and plate numbers of the microplate. A coordinate marker was prepared by mixing the two primers, AFLP was performed on these with the same primer pair using genomic DNA as a control, and then electrophoresed using this system to amplify the same band as the control. are, row,歹¹ J, the plate coordinates, clones having the desired band without resorting to hybrida I See Chillon can be identified.

9. Making a contig covering the whole genome of an organism

If it is possible to clarify the adjacency of all the constituent clones based on the genomic library of a certain organism using the BAC, etc., it is necessary to connect the individual contigs to develop a contig (physical map) that covers the entire genome. Can be. The physical map created in this way corresponds to a so-called reproduction of the genome structure of the organism, and by this completion, the isolation and identification of important functional genes does not necessarily have to wait for the entire genome sequence. It's very easy to do, and you'll be able to pick up the organism's genome. In fact, once the whole genome contig is completed, the work of the subsequent genome sequence can be almost completely automated. However, until now, the preparation of whole genome contigs could only be handled by a large research group with dozens of people. However, if the present invention is used, rice With a genome size (approximately 500MB), it is possible for one person to create a contig that covers the entire genome in about one year.

Until now, the major reason that whole-genome contigs were difficult was that it was difficult to obtain specific markers that mark each clone constituting the genomic library. In the present invention, in combination with AFLP, very specific bands labeled with two parameters, namely, the three base sequences at both ends and the number of bases (length), are obtained at one time in the number of 30-50 in one lane. be able to. All of these specific bands can correspond to the constituent clones of the genomic library, and the distribution density of the band can be made sufficiently smaller than the length of the BAC clone, so that clones sharing the same marker can be used. It can be said that connecting is not difficult. At this point, in order to obtain a nearly one-to-one correspondence between the AFLP band and the library clone, the genome library of the whole genome, which is usually several genomes or more, is divided into several sublibraries equivalent to about one genome. . Furthermore, the clones that make up the sublibrary are uniquely determined if the row, column, and plate numbers of the microplate are the coordinates, so the row, column, and plate numbers are the same axes as the coordinate axes. A small amount of DNA is collected from a clone with coordinates and mixed to make a coordinate sample that represents the coordinates of the row, column, and plate axes. The genomic DNA is subjected to the genomic scanning method of the present invention by using the genomic DNA as a バンド, and when the AFLP pattern is compared at both ends of the coordinate lane with the reference genomic DNA as the 鍀, a specific band having the genomic DNA as 錶 is obtained Corresponding clones can be easily detected from the sublibrary. Assuming that there are two or n-number of corresponding clones subgroups, although eight or n ³ pieces of clone 2 ³ will correspond with the band, in this case it takes out the 8 clones If the second electrophoresis is performed by the genome scanning method, two truly corresponding clones can be identified. A specific method for preparing a coordinate sample of the sublibrary is shown in Example 5. In this way, if the library is about 120 kb in size and the genome size is about the same as the blast fungus (about 40 MB), the sublibrary equivalent to about 1 genome is about 300 clones, and 8 rows, 6 columns, 6 and a half. Can be placed on a plate. In this case, even if 20 control sample lanes and 22 control lanes (22 lanes in total) are run, two gels can be used to migrate a sublibrary equivalent to 6 genomes, that is, a whole genome library. That is, if it is possible to identify and respond to approximately 60 bands of 2 primer pairs in one run, 1500 bands can be processed in 25 runs. This is a density of 40 MB / 1500 lines = 27 kB / line, which is equivalent to the identification of 4.4 bands on average for a BAC clone with an average size of 120 kB. Considering the overlap, it can be seen that almost all genomics are expected to be covered by this (see Example 5).

For a library equivalent to 6 genomes with an average insert size of 150 kB, even for a rice genome size (450 MB), a matrix of 16 rows, 12 columns, and 16 x 2 plates using 32 plates with almost 1 genome equivalent By preparing the above, it is possible to perform electrophoresis for six sublibraries at one time, and about 25 bands are identified at one time. 5000 runs were identified in 200 runs, corresponding to a density of 450 MB / 5000 = 90 kB / row, so a 150 kB average clone from a library with 6 times the degree of duplication had a longer contig. It is considered that the density is sufficient to produce. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a front view and a top view of a main body of a standard electrophoresis apparatus used for a genome scanning method. Four 18 cm square gels can be run simultaneously. One gel has a comb for carrying 66-68 specimens with a width of 1 millimeter. A well for sample application is made. 2-4 lanes are used for flowing molecular weight markers, etc. With 4 gels, 256 samples can be run at a time. Efficient PCR-amplified samples can be applied to a gel by using 8 micro-mouth pipes.

FIG. 2 is a diagram showing a gel plate stopper part of a standard electrophoresis apparatus used for the genome scanning method.

FIG. 3 is a diagram showing a standard electrophoresis apparatus used for the genome scanning method. Figure 4 is a photograph showing a finished product of a standard electrophoresis apparatus used for the genome scanning method.

FIG. 5 is an electrophoretogram showing primary screening using a genome scanning method for candidate markers near the apathogenic gene avrPib of the rice blast fungus obtained by bulk analysis. Genomes combined with AFLP in the parent strains avrPib—, +, bulk avrPib—, ju, F1 strain avrPib—6 strains, and +6 strains for the primer pairs A, B, C, and D, each having four types of nearby marker candidates. Scanning analysis was performed. The triangles in the figure indicate bands that are candidates for a marker. Primary screening for A, B, and C all showed genotype and cosegregation as expected, but recombination was observed for every eleven strains for D (closed triangles). Note that 5-60 bands were obtained for each primer pair. One gel can scan 3 to 4 000 bands, and 4 gels can be run at a time, so 12 to 16,000 bands can be scanned. The number of selected nucleotides in the primer pair used here was 3 bases on the EcoRI side and 1 base on the Mspl side. The polymorphism rate between the hybrid strains used here was about 5%, and the total genome of the blast fungus was about 500 CM.Therefore, about 500 polymorphic bands were obtained with 256 primer pairs, and 1 CM / Marker density covers the entire genome.

Figure 6 shows the results obtained by mapping the non-pathogenic gene avrP ib, a non-pathogenic gene of rice blast fungus, obtained by the RAPD method and the genome scanning method using 125 F1 strains by each method. 3 shows a detailed map of the vicinity of the obtained gene. As shown in Table 1, 700 primers were used in the RAPD method, and 251 primer-pair PCR was used in the genome scanning method. We searched for neighbors. The search and mapping of markers by the RAPD method took three months, while the genome scanning method was completed in January. The number of bands counted was limited to very clear ones, so they are quite small. An: A marker obtained by the genome scanning method. (Rn): The power obtained by the RAPD method.

Fig. 7 is an electrophoresis photograph showing an example of bulk analysis for searching for a marker adjacent to be-3, which is a cellulase mutant mutant of rice. The set of 4 lanes is from left to right: mutant parent (Mil: japonica), mutant (recessive) homo bulk, wild-type (dominant) homo bulk, wild-type parent (Kasalath: indica). Arrows indicate candidate markers near the gene where a clear difference between the bulks is observed. Even in the case of a clear band alone, 20 to 30 lines are counted per lane. If a slightly narrow band is included, the theoretical value of around 50 lines is confirmed in one lane.

Fig. 8 shows the crossing of azum wheat (Article 6) X Kanto medium-grade gold (Article 2) and backcrossing with Azum wheat (Article 6) for 7 generations in order to search for genes that determine the bisexuality of oats. This is a photograph comparing genomic scanning of a line that was established as a two-line near-isogenic line with a background of azum and a return parent, azum, in which a line exhibiting striation was selected. A search was made for differences between a pair of 16 primers in 32 lanes, but there were very few differences between the two primers, and these were candidates for polymorphic bands in a region strongly linked to the bisection gene.

FIG. 9 shows an example of SCAR (Sequence Characterized Amplified Region) marker obtained by isolating a band specific to “Akitakomachi”. Primer created by inversion. B is an electrophoresis photograph showing bands amplified by PCR from 10 major varieties of nucleic acids using the primer of A. Specific bands are amplified only in Akitakomachi. control 1 is a specific band isolated for sequencing as a template.

FIG. 10 shows a method for directly selecting a clone corresponding to a specific band from a genomic library using a genome scanning method. Here, blast fungus (genome size A library of 6 genomes with an average insert size of 120 kB (40 MB) was divided into six sublibraries of almost one genome, and clones corresponding to the genome scanning band were identified from each library. I have.

In Fig. A, the clones indicated by black circles are displayed as row C, column 4, and a plate. Coordinates A coordinate sample representative of line C is made by collecting 10 ng of the DNA of the clones in all columns on line C of all half plates. In Fig. B, one sub-library consists of a coordinate sample for eight rows, six columns, and six half plates, and a target genomic lane, for a total of 22 lanes. Six genomes can be run simultaneously on two gels, and the corresponding clone is identified from the coordinates that show the band that matches the control band.

FIG. 11 shows an example in which a genetic map of wheat was prepared by electrophoresis on a 15-lane type gel, which is the predecessor of the genome scanning method. Out of a total of 272 markers, 227 AFLP markers were mapped in this way. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Non-pathogenic gene of rice Nucleic acid near the avrPib gene Precision mapping

Regarding the non-pathogenic gene avrPib of the blast fungus corresponding to the rice resistance gene Pi-b, we attempted to search for a nucleic acid marker in the vicinity and determine the genetic distance between the obtained marker and this gene Was. avrPib is a non-pathogenic gene of blast fungus corresponding to the rice resistance gene Pi-b.Rice resistance is established by the recognition of this gene product by the rice Pi-b gene. Mutation in the avrPib gene will disrupt resistance to Pi-b. Therefore, the cause of the mutation in the strain that caused the resistance collapse by isolating this gene, the resistance gene product and the non-pathogenic gene product We need to find out how they interact. By crossing with blast fungus that infects Pi-b isolated from the area where resistance collapse actually occurred, precise mapping of non-pathogenic genes was compared between the RAPD method, which is a representative of the conventional method, and this method did.

A strain containing avrPib (which cannot infect rice with Pi-b) and a strain without avrPib (infecting rice with Pi-b) were crossed to obtain a large number of strains of the F1 generation. . After inoculating these F1 strains into rice plants having P i -b and examining the presence or absence of avrP ib in each strain, genomic DNA was obtained from more than a dozen strains each of the avrPib holding group (+) and the deletion group (-). Bulks were prepared, and AFLP analysis was performed using 256 types of primer pairs (64 x 4 types = 256 types) using 4 types of samples including 2 parent strains and 10 types of each bulk type. Since the genome size of blast fungus is about 40MB, which is about 1/10 of rice, the genome can be covered with about 1/16 of the primer pair of rice.

For comparison, the results were compared with the results of amplification using about 540 primers by the RAPD (Random Amplified Polymorphic DNAs) method. In the RAPD method, for example, 144 lanes can be run simultaneously at the same time on a large submarine gel, but in the bulk method, 540 x 4 = 2160 lanes need to be run, and over 15 days, a highly stable and obvious The band alone compared 1860 bands between bulks and found about 100 parental polymorphism bands.

On the other hand, in this method, about 5700 bands could be compared and searched in 4 days, and 304 polymorphic bands were found (Table 1). This indicates that the efficiency of this method is 11 times that of the RAPD method (5700/1860 x 15/4).

table 1

Used primers Total Polymorphic bands Average Percentage of (pairs) bands bands / polytnorophism obtained primer (pair) (V.)

Total Linked to avrPib

(Closely linked)

RAPD 539 1861 101 10 (4) 3.5 5.4

AFLP 251 5710 304 86 (41) 22.7 5.3 Among these polymorphic bands, the candidate genes near the gene that showed a clear difference between dominant homozygous and recessive homozygous in the bulk method were 6 and 41 in RAPD and AFLP, respectively. These bands were first tested using 12 actual F1 strains and subjected to primary screening (Fig. 5). Furthermore, we increased the number of F1 strains, and finally made a precise map around avrPib by testing with 125 F1 loaf strains (Fig. 6). The RAPD method showed that 2 bands and the present method showed 12 bands were actually within 20 CM of the target gene avrPib. The band closest to the target gene was 5.3 CM obtained by the RAPD method, whereas the band obtained by this method was much closer to 1.8 CM. Since 1 CM is equivalent to about 80 kB for blast fungus, this distance can be said to be a distance that can be sufficiently transferred to physical map creation.

[Example 2] Precise mapping of one of the rice camillaz mutant genes, be-3, a mutant that is unable to synthesize cellulose required for secondary thickening of the cell wall of rice bar (force myraz: brittle culm) A genome scan was used to search for nucleic acids near the causal gene of be-3, one of the genes. As a parent strain of the cross for F2 analysis, a cross with a japonic a-type be-3 mutant: Mll and an indica-type wild-type Kasalath was used. Analysis of up to F3 generations identified 10 mutant homozygotes and 8 wild-type homozygotes, and combined the genomic MAs with the parental strain and performed bulk analysis by genomic scanning combined with AFLP Was served. After double digestion of each nucleic acid with EcoRI (6 bases recognition) and Msel (4 bases recognition) two enzymes, a bimer with three base selection nucleotides adjacent to the cleavage site of each enzyme For 1430 combinations, a genomic scanning method based on the no and rirek analysis was applied. Figure 7 is an example. As a result, 97 nearby marker candidates were obtained, of which 50 could be effectively used to detect recombinant markers from recessive homologous individuals. For each marker candidate, the markers were narrowed down by performing F2 analysis using 10 recessive homologous individuals (20 chromosomes). As a result of analyzing 64 chromosomes in an individual, one marker (with a distance of 0 CM from the gene) co-separated from the target gene was found (Fig. 7).

[Example 3]

For organisms with a very large genome size, such as wheat (5.5 GB), when the present invention is applied by AFLP, when the number of bases selected by the primer on each side of the genomic fragment is 3, rice (450 MB) If DNA is electrophoresed in a double-stranded state under non-denaturing conditions, such as a small genome size, a clear band will not always be obtained. In this case, increasing the number of selective primers is one possible solution.However, even if three selective techniques are used, the sample buffer may be added under denaturing conditions in which 6-8.5 M urea is added to the gel. Also, 50% formamide is added to the DNA, and heat denaturation is performed at 90 ° C for 3 minutes immediately before the electrophoresis. As a result, sufficient resolution of bands can be obtained when DNA is electrophoresed as a single strand.

Fig. 8 shows the search for genes that determine the bisexuality of barley. In order to search for genes that determine the bisexuality of barley, crossing with Azumamugi (Article 6) X Kanto Nakasei Gold (Article 2) and backcrossing with Azumamugi (Article 6) were repeated for seven generations. We selected strains that showed sex and compared the two established near-isogenic lines with azum in the background with the returned parent azum by the genome scanning method. A search was made for differences between 16 primer pairs in 32 lanes, but the differences between the two were extremely limited, and these are candidates for polymorphic bands in a region strongly linked to the double-stranded gene.

Under these electrophoresis conditions, an average of about 58 bands were identified in wheat. Since the average number of bands obtained by AFLP of rye using a large sequencer gel is about 88 (Castil ioni et al. 1998 Genetics 149: 2039-2056), about 67% of the bands are identified. It will be. Where large gels can be processed only with an efficiency of about 32 lanes per gel per day, the present invention can process 256 lanes per day, so that 5 to 6 times the efficiency can be obtained.

[Example 4] Isolation of identification bands of rice varieties identified by genome scanning method and design of specific primers by sequence analysis We developed a SCAR marker to easily identify commercial rice varieties, and attempted to construct a system that allows anyone to easily identify varieties by performing PCR on the spot.

Using the genome scanning method (AFLP), bands that identify the 10 major rice varieties commercially available were searched using 55 types of primers. This 1 = diet only takes 2 days. A number of bands suitable for discrimination were obtained, but one of the bands specific to Akitakomachi was electrophoresed in a wide lane and then stained with a fluorescent dye (vistra green) and cut out. The DNA was extracted by crushing in a TE buffer and amplified by PCR using primers for AFLP. Insert the PCR amplification product into an appropriate plasmid, introduce it into E. coli, and confirm that the desired insert is contained in the plasmid obtained by culturing and amplifying the bacteria with restriction enzymes, and analyze the sequence. A primer was designed to specifically amplify the target band (FIG. 9A). Using these primers, PCR amplification was performed using DNA obtained from the main 10 varieties as templates, and it was confirmed that the specific band was amplified only in “Akitakomachi” (Fig. 9B).

[Example 5] Method for selecting a clone of a library corresponding to a nucleic acid marker Normally, when a clone corresponding to a specific band obtained by the present method is identified from a genomic library, as in Example 3, Using the SCAR marker obtained in the above, colony hybridization is performed on a high-density membrane carrying the library to select positive clones. Using all methods requires too much labor and cost, and the internal sequence of the isolated band is not always unique.

A method for directly selecting a library clone corresponding to a specific band by a genome scanning method without isolating the marker-one band may be as follows. First, the DNA of a genomic clone such as BAC is extracted from all the library clones using a plasmid extractor to prepare a plate of DNA having the same sequence as the plate of the original clone. Next, divide the whole genome library to It is a set of size sub-libraries. This ensures that on average only one clone per sublibrary corresponds to a particular band. In addition, 10 ng of DNA is collected from clones with the same coordinate number using the row, column, and plate numbers as the coordinates from several microplate clones that constitute each sublibrary, and correspond to each coordinate. Make a coordinate sample. For example, from the clone in the third row, all 10 ng of DNA is collected and collected, regardless of the plate number or column number, and used as a coordinate sample on the third row. Similarly, create coordinate samples for all coordinates, and apply the same genomic scanning method for each coordinate sample and the control whole genome sample. To prepare the coordinate sample MA, even without extracting the MA of all clones in the genomic library, the culture of each clone, which had been increased to about 2 ml, was mixed in small amounts in each row, column, and plate, and the coordinate sample and MA were extracted. After that, DNA may be extracted from the mixed culture. If a band, column, or plate is found that matches the band of interest in the lane amplified from the control whole genome, that number will be the 3D coordinates of the desired clone, and that clone will be picked up from the sublibrary. it can. If there are many candidate clones in the sub-library, they should be collected and re-run the genome scanning method for final determination together with the control (Fig. 10).

This method is most effective when all the bands obtained by a large number of genome scanning methods correspond to one library to create a contig of the whole genome so as to cover the constituent clones of the genomic library. is there. This makes it easy for one person to create a whole genome container.

In Figure 10A, one half of the genomic library of blast fungus (40 MB) (average 120 kB; equivalent to 6 genomes) was divided into six subgenomic libraries, each of which corresponds to almost one genome. It shows how to identify a specific clone from one subgenomic library consisting of one full plate). For the 1 subgenome library, the first row of row coordinates is the first row of six half-plates. DNA samples are collected in 10 〃g increments from all of the loans (6 rows x 6 half-plates = 36 clones) and used as coordinate samples representing the first row. In other words, in one subgenomic library, each coordinate sample of 8, 6, and 6 is used as coordinates representing the row, column, and plate axes, and a total of 20 coordinate samples, 8 x 6 x 6 = 288 for the entire subgenome library The clone can be identified.

As shown in Fig.10B, 20 lanes using the 20 coordinate samples as a template and 2 lanes using the whole genome of the template as a template were simultaneously swept by the genome scanning method, so that the control was easily performed in 22 lanes. Clones corresponding to the obtained bands can be identified from one subgenomic library.

Since one gel (66 lanes) can simultaneously run three subgenomic libraries, two gels can cover the entire genome and six sublibraries. That is, considering one AFLP with one primer pair, it is possible to search for clones corresponding to about 25-40 bands on two gels for a whole genome library equivalent to 6 genomes. Since the standard genomic scanning method uses four gels, clones corresponding to the 50-80 bands obtained by two primer pairs in one swim can be similarly identified from the whole genome library.

Thus, even if conservatively estimated, it is possible to associate about 50-80 bands of the two primer sets to the clone in one electrophoresis, and about 1,000-2,000 bands in 25 electrophoresis. Band can be associated with the genome. Considering that the genome size of the blast fungus is 40 MB, the average density of the bands in the genome is 40 MB ÷ 1,500 = 27 kB / line, assuming that 1,500 bands are obtained, and the average of 120 kB is obtained. This means that the clones will have an average of 4.4 bands and that the clones have an average of 6 duplications, which is considered to be too high for a genomic contig. As a result, a contig of a whole genome library consisting of 6 genomes will be completed in about one month if only the BAC plasmid is prepared. If the automatic plasmid extractor can be fully used, it is possible to prepare 18 plates of plasmid in about two weeks. Industrial applicability

By using the method and the electrophoresis apparatus of the present invention, for example, it has become possible to search, identify, and acquire nucleic acid markers for the following purposes extremely easily and with high efficiency.

(1) Important functions · Development of polymorphic markers that mark single genes that control traits using polymorphisms of nucleic acids in the vicinity.

In this case, it is not necessary for the organism having the target gene to have a genomic map consisting of existing markers.

② Identification and isolation of clones near the target gene in positional cloning

An important function: When a single gene that controls a trait is isolated and cloned by positional cloning based only on its chromosomal location, a marker near the chromosome is searched for and the BAC (Pacteria Provide a marker for lifting a clone in the vicinity of a gene from a genomic library such as an artificial chromosome). In this case, it is not necessary that an organism having the target gene has a map composed of existing markers.

③ Genetic analysis, making high-density maps

Along with providing a large number of nucleic acid markers for creating a genetic map of an organism with high efficiency, the linkage analysis in F2 or RI strains will be performed with high efficiency, and a high density map will be generated with high efficiency. This facilitates the analysis of quantitative traits (QTLs) based on the contribution of multiple genes.

④ Breed identification Highly efficient marker bands for identifying rice and other foods and seed varieties on the market. Furthermore, the obtained discriminating band is isolated and cloned, and from the sequence analysis, primers for SCAR (Sequence Characterized Amplified Region) analysis are designed. The use of such an SCM marker makes it possible to identify the type in a short time on the spot. In addition, by converting the vicinity marker that marks a specific gene into an SCM, it is easy to use as a primary tool in breeding, etc., and it is also useful to catch BAC clones near the gene.

の Creating a contig covering the entire genome of an organism

By concatenating clones constituting a genomic library of an organism, a contig covering the entire genome can be easily prepared. That is, after dividing a genomic library consisting of several genomes into one sub-library equivalent to about one genome, the rows, columns, and plates of each set of microplates constituting each sub-library are divided into x, Create coordinates using the y- and z-axes, collect all the DNAs of the clones that are orthogonal to each coordinate, and prepare a coordinate sample to form a 錡. By running this together with a normal whole genome MA type II control along with the genome scanning method, clones corresponding to each band found in the whole genome lane can be identified (FIG. 10). If bands are found at a density of about 1 band / 20-50 kB, a whole genome contig consisting of several clones on average can be completed.

Claims

The scope of the claims

1. A method for electrophoresing nucleic acids,

(a) An electrophoresis apparatus capable of simultaneously carrying out electrophoresis by equipping a plurality of gel plates of 10 to 30 cm square and simultaneously carrying out electrophoresis of 32 or more nucleic acid samples per gel plate Electrophoresing a nucleic acid sample using

(b) detecting a nucleic acid band on the gel after electrophoresis.

2. The method according to claim 1, wherein the electrophoresis is performed using a discontinuous buffer type gel.

3. The method according to claim 1, wherein the nucleic acid sample is single-stranded DNA prepared by dissociation of double-stranded DNA by denaturation treatment, and electrophoresis is performed using a denaturing gel.

4. The method according to claim 1, wherein the detection of the nucleic acid band on the gel is performed by fluorescent staining or silver staining.

5. The method according to any one of claims 1, 2, and 4, which is performed to detect a genomic DNA polymorphism between test subjects.

6. The method according to claim 3, which is carried out to detect a genomic DNA polymorphism between test subjects.

7. The method according to claim 5, wherein the nucleic acid sample is a DNA fragment amplified by the AFLP method.

8. The method according to claim 5, wherein the nucleic acid sample is a heterologous double-stranded DNA.

9. A method for preparing a DNA fragment exhibiting a polymorphism, comprising a step of isolating a DNA fragment exhibiting the polymorphism detected in the method according to any one of claims 5 to 8 from a gel.

10. A DNA fragment showing polymorphism among test individuals isolated by the method according to claim 9.

11. The method according to any one of claims 1 to 8, which is performed for performing a genetic analysis.

12. The method according to claim 11, wherein the genetic analysis is F2 analysis, RI (recombinant imbred) analysis, or QTL (Quantitative Traits Loci) analysis.

13. The method according to any one of claims 1 to 8, which is performed for creating a genetic map of an organism.

14. A genetic map of an organism created using a genomic DNA band showing a polymorphism detected by the method according to claim 13 as a marker.

15. A method for selecting a clone corresponding to a specific nucleic acid band on a gel detected by the method according to any one of claims 1 to 8 from a genomic DNA library,

(a) dividing the genomic DNA library of a particular organism into a plurality of sub-libraries each of which is smaller than one genome of the organism;

(b) The process of assigning the row, column, and plate numbers of the sublibrary to all the clones included in each sublibrary (where the rows, columns, and plates are designated as coordinate X, coordinate Y, and coordinate Ζ, respectively). Do),

(c) A clone representing a particular row of all plates (coordinates X clones), and a clone representing a particular column of all plates (coordinates Υ clones), and one particular sublibrary on a particular plate Collect all the clones (coordinates Ζ clones), extract DN に separately and use them as coordinate samples, prepare genomic DNA prepared from the organism as a control, request the coordinate samples and controls side by side Electrophoresis by the method described in any one of Items 1 to 4, and a step of detecting a band,

(d) The band having the same mobility on the gel as the band of the target nucleic acid in the control is identified in each of the clone groups at the coordinate X clone group, the coordinate Y clone group, and the coordinate Z clone group. A step of determining whether the coordinate is a clone,

(e) selecting a clone corresponding to the determined three-dimensional coordinates from the sub-library.

16. The method of claim 15, which is performed to create a contig covering the entire genomic DNA of a particular organism.

17. Electrophoresis device for electrophoresing nucleic acids, which can be equipped with multiple gel plates of 10 to 30 cm square at the same time to perform electrophoresis, and more than 32 per gel plate An electrophoresis apparatus capable of simultaneously electrophoresing nucleic acid samples.