JP2002540799A - A new type of transposon-based genetic marker - Google Patents

Abstract

  (57) [Summary] The present invention relates to the use of DNA primers homologous to MITE in a method for detecting polymorphisms in a nucleic acid sequence. The method comprises amplifying a nucleic acid sequence using a first primer homologous to MITE in combination with another primer that is homologous or not homologous to MITE, separating fragments of the amplified nucleic acid sequence, and Analyzing the fragments obtained with respect to the reference fragment obtained from amplifying the nucleic acid sequence using primers homologous to MITE to detect polymorphisms therein. DNA primers homologous to MITE may also be used for genotyping, fingerprinting, mapping or cloning according to the invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION (a) Field of the Invention The present invention relates to a first primer having a DNA sequence homologous to a small inverted repeatable transposable element (MITE) and a second primer identical to or different from the first primer. And a method for determining the genotype of a nucleic acid using amplification by a primer pair. The present invention relates generally to the use of MITE primers in fingerprinting or linkage studies. (B) Conventional technology McClintock (McClintock B. 1946. Maize)
genetics. Carnegie Inst. Wash Yearbook
45: 176-186; and McClintock B.C. 1947. Cyt
Ogenetic studies of maize and neuros
pora. Carnegie Inst. Wash. Yearbook 46:
146-152. ), The identification of genes of the novel metastatic element system (family) was followed by the identification of plants (Peterson PA.
1986. Mobile elements in maize. Plant
Breeding Reviews 4: 3-122. ) As well as a popular area for genetic research in other organisms. This involves characterization of the molecules of transposable elements and exploitation of these elements as a means of gene identification and isolation, especially after cloning of the white locus by the copier retrotransposon in Drosophila (Bingham PM, R. et al. Lewis and G.
M. Rubin 1981. Cloning of DNA sequence
s from the white locus of D.S. melanoka
ster by a novel and general method. C
ell 25: 693-704. ), And the transposable element Ac (P
ohlman R.S. F. , N .; V. Fedoroff and J.M. Messi
ng 1984. The nucleotide sequence of t
he maize controlling element Activat
or. Cell 37: 635-643. ) And En / Spm (Perair
a. , Zs. Schwarz-Somer, A .; Gierl, I .; Ber
tram, P .; A. Peterson and H.M. Saedler 1985
. Genetic and molecular analysis of
he Enhancer (En) transportable element
nt system of Zea mays. EMBO J.C. 4: 17-25
. ). Since then, research on metastatic elements has become a major center in biological science.

As in other areas of biological research, the identification of metastatic elements has been facilitated by modern computer technology. Bureau et al. (Bure
au T.A. E. FIG. , P. C. Ronald, andS. R. Wessler 19
96. A computer-based systematic probe
y reviews the predominance of small
inverted-repeat elements in wild-type
erice genes. Proc. Natl. Acad. Sci. 93: 8
524-8529. ) Adopted this approach to identify a vast number of novel families of metastatic elements. These elements are terminally inverted repeats (TI
(Rs) is similar to traditional DNA-mediated transposable elements (
Contrary to retro elements transferred by RNA intermediates, Boeke J. et al. D. , D
. J. Garfinkel, C .; A. Styles and G. R. Fink
1985. Ty elements transpose through
an RNA intermediate. Cell 40: 491-500.
). However, unlike metastatic elements characterized by classical genes,
These elements are small in size and apparently do not show coding capacity. These elements are small inverted repeating transposable elements or MITs
(Bureau et al., Supra).

[0003] Restriction fragment length polymorphism (RELP) technology (Bostein D., R. White
, M .; Skolnick and R.S. W. Davis 1980. Const
fraction of a genetic link map in
man using restriction fragment length
h polymorphism. Am. J. Hum. Genet. 32: 314
-331. ) Has been substantially advanced since its introduction as a molecular mapping tool, and other novel techniques, such as randomly amplified DNA polymorphisms (RAPD, Welsh J. and MM), have been developed.
cClellland 1990. Fingerprinting genome
s using PCR with arbitrary primers. N
ucleic Acids Res. 18: 7213-7218. ; Willi
ams J .; G. FIG. K. , A. R. Kubelik, K .; J. Livak, J. et al. A
. Rafalski and S.A. V. Tingey 1990. DNA po
lymorphisms amplified by arbitrary p
rimers are useful as genetic markers
. Nucleic Acids Res. 18: 6531-6535. ), And length polymorphisms in the amplified fragment (AFLP, Vos P., R. Hogers,
M. Bleeker, M .; Reijans, T .; van de Lee, M .; H
ornes, A .; Fritters, J.M. Pot, J.M. Peleman, M .; K
uiper and M.S. Zabeau 1995. AFLP: a new t
echnique for DNA fingerprinting. Nucl
eic C @ Acids Res. 23: 4407-4414. ). Recently adopted technology using retro elements (Sinn
et D. , J. et al. -M. Deragon, L .; R. Simard and D.S.
Labuda 1990. Aluminophs-human DNA poly
morphisms detected by polymerase cha
in reaction using Alu-specific prime
rs. Genomics 7: 331-334; and Nelson D. L.
, S .; A. Ledbetter, L .; Corbo, M .; F. Victoria,
R. Ramirez-Solis, T .; D. Webster, D.A. H. Ledb
etter and C.I. T. Caskey 1989. Alu polymer
race chain reaction: A method for rap
id isolation of human-specific DNAs
equipments from complex DNA source. Pro
c. Natl. Acad. Sci. 86: 6686-6690. ) And simple sequence repeats (SSRs) (Litt M. and JA Luty 198).
9, A hypervariable microsatellite rev
earled by in vitro amplification of a
dinucleotide repeat without the card
iac muscle actin gene. Am. J. Hum. Genet
. 44: 397-401; Tautz D.C. 1989. Hypervariab
ility of simple sequences as a generator
al source for polymorphic DNA marker
s. Nucleic Acids Res. 176463-6471; and W
eber J. L. and P.S. E. FIG. May 1989. Abundant
class of human DNA polymorphisms whi
ch can be typed using the polymerase
chain reaction. Am. J. Hum. Genet. 44:38
8-396. ) Has provided a new generation of stages of genome mapping and fingerprinting tools.

[0004] Restriction fragment length polymorphism (RELP) marker methodology involves cutting genomic DNA with a restriction enzyme, separating the DNA fragment by electrophoresis, and transferring the separated DNA fragment to a nylon membrane support. Thereby obtaining an image of the gel on a solid support that can be used for hybridization experiments with known DNA. The known DNA sequence can be the sequence of a cloned genomic or cDNA or a specific PCR product. This DNA sequence (probing sequence) is labeled with radioactive, fluorescent or chromogenic nucleotides. Hybridization results can be observed by exposing the solid support to an X-ray sensitive film, or directly on the support if chromogenic nucleotides are used to label the probe. One or a few DNA bands are often observed depending on the origin of the probing sequence. Restriction fragment length polymorphisms are visualized as differences between the banding patterns of different genotypes, and reflect the distribution of given restriction enzyme cleavage sites.

[0005] The methodology of markers for random amplified polymorphic DNA (RAPD) is
Consists of a short DNA sequence of 10 nucleotides used as a primer to steer a PCR reaction using NA as a template. The composition of the nucleotides of the oligonucleotide primers is selected arbitrarily without any reference to the DNA sequence present. PCR products are visualized directly after agarose gel electrophoresis.
Generally, 1 to 15 amplified DNA fragments can be observed as amplification products of the eukaryotic genome. Polymorphisms are detected directly on agarose gels as differences in amplification patterns between genotypes and reflect single nucleotide changes and insertions / deletions in primers.

[0006] Markers for amplified fragment length polymorphism (AFLP) can be obtained by cutting genomic DNA with a restriction enzyme and converting the resulting genomic DNA fragment to an adapter sequence (the restriction fragment used to digest the genomic DNA). (A short double-stranded DNA sequence having at one end the same sequence site generated by the enzyme) and performing a PCR reaction using, as a primer, an oligonucleotide homologous to the adapter sequence. The results of the amplification are visualized directly on an acrylamide gel after staining as several (up to 60) DNA fragments. Polymorphism is observed as a difference in the presence / absence of a particular amplified DNA fragment in another genotype, and REL
Like P, it reflects the differences in the distribution of given restriction sites, but with a subset of genomic DNA.

[0007] Marker methodology for simple sequence repeat (SSR) is based on simple DNA sequence repeats (eg, (TA) n, (CAGA) n, (GA) n, etc., where "n" is typically between 5 and 18). Variability) as a probe for identifying genomic clones from gene libraries of organisms having these simple sequence motifs. The isolated clone is then sequenced, and a pair of DNA primers surrounding the SSR is designed for amplification of the SSR and the surrounding DNA sequence. Polymorphisms are observed as one or very few amplified DNA fragments that vary by one or a few nucleotide differences in different genotypes and reflect differences in the number of simple sequence repeats ("n").

[0008] DNA markers based on retro elements and other large repetitive elements consist of designing primers surrounding the element, and polymorphisms can occur when the element is present or absent of another genotype. Is found.

[0009] There are other types of DNA markers, which are combinations of the above DNA marker types. For example, when a PCR product is cleaved by a restriction enzyme after PCR amplification, CAPs are cleaved amplified polymorphic DNA. Primer pairs can be designed from repeat elements and AFLP primers, or from another repeat element.

It would be highly desirable to provide new and widely used nucleic acid sequences for use in linkage studies and fingerprinting studies. It would also be highly desirable to provide a method for detecting polymorphisms in eukaryotes using this novel and widespread nucleic acid sequence. SUMMARY OF THE INVENTION One object of the present invention is to provide new and widely used nucleic acid sequences for use in linkage studies and fingerprinting studies.

[0011] Another object of the present invention is to provide a method for detecting polymorphisms in eukaryotes using this novel widespread nucleic acid sequence. According to the present invention, there is provided a method for detecting a polymorphism in a nucleic acid sequence of interest.
The method comprises the steps of: a) converting the nucleic acid sequence of interest to a first primer homologous to a small inverted repeatable transposable element (MITE), a fragment or derivative thereof, and the first primer comprising the nucleic acid of interest. A second anneal to the MIT when present in the sequence
Amplifies with a primer, except that the second primer is identical or not identical to the first primer and is or is not homologous to the MITE sequence; b) the interest of interest amplified in step a) Isolating a fragment of the above nucleic acid sequence; and c) obtained by amplifying a nuclear sequence using at least one primer to detect a difference in nucleic acid sequence between the fragment obtained in step b) and a reference. With respect to the reference fragment, the fragment obtained in step b) is analyzed, whereby the difference is indicative of a polymorphism in the nucleic acid of interest.

Also, according to the present invention, there is provided a method for determining a genotype of a eukaryote. The method comprises the steps of: a) amplifying a nucleic acid sequence of interest with a first primer homologous to a small inverted repeatable transposable element (MITE), a fragment or derivative thereof, and a second primer, provided that A first primer anneals to the MITE when present in the eukaryotic nucleic acid sequence and a second primer is or is not identical to the first primer and is homologous to the MITE sequence. B) separating the fragment of the nucleic acid sequence of interest amplified in step a); and c) the fragment obtained in step b) and a reference nucleic acid sequence from the eukaryote Whereby the identity of the fragment of step b) and the reference nucleic acid sequence is indicative of a eukaryote having said nucleic acid sequence.

Further, according to the present invention, there is provided a method of fingerprinting a eukaryotic organism. The method comprises the steps of: a) amplifying a nucleic acid sequence of a eukaryotic organism with a first primer homologous to MITE, a fragment or derivative thereof, and a second primer, provided that the first primer has a MIT sequence. Specific and the second primer is identical or not identical to said first primer and is or is not homologous to the MITE sequence; b) from amplifying the nucleic acid sequence of step a) The fragments obtained are separated, whereby the fragments thus separated are representative of eukaryotic organisms.

Preferably, the step of amplifying is performed by a PCR technique. The first primer is derived from the consensus sequence from the MITE element. More preferably, the first primer is a Tourist, Stockaway, Barfly or Mari.
has a nucleic acid sequence derived from the consensus sequence from ner.

[0015] Most preferably, the first primer comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:
, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 are 3
0, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

[0016] The second primer is optionally a MITE sequence-specific primer, a SSR sequence-based primer, a retro element sequence-based primer RELP-directed cloned nucleic acid sequence-based primer, a random genomic sequence-based primer and It is a primer selected from the group consisting of primers based on gene sequences.

Also, according to the present invention, to track the progeny of eukaryotic organisms, to measure eukaryotic hybridisation, to identify variations in linked phenotypic characteristics in eukaryotic organisms To provide for the use of a polymorphism, such as according to the method of the invention, to identify individual progeny from a cross, wherein said progeny is from a parent donor and / or recipient parent, or It has the desired genetic contribution as a genetic marker for constructing a genetic map.

The method of the present invention may be used to isolate genomic DNA sequences surrounding a gene coding or non-coding DNA sequence. The genomic DNA sequence surrounding the gene coding DNA sequence is preferably a promoter sequence or control sequence.

Further, according to the present invention, a nucleic acid fragment or a derivative thereof obtained by amplifying a eukaryotic nucleic acid sequence with at least one primer homologous to MITE is used as a probe on the nucleic acid sequence. Provided for use.

The nucleic acid fragments or derivatives thereof may be used for marker-assisted selection (MAS), map-based cloning, hybrid assays, fingerprinting, genotyping, and allele-specific markers.

The eukaryote or eukaryotic organism is preferably a plant, animal or fungus. Further according to the present invention there is provided a method of genomic mapping, comprising the steps of: a) fractionating a genomic of a eukaryotic organism; b) cloning the fractionated genomic into a vector; The thus cloned vector is tested by amplifying the DNA in the cloned vector using first and second primers homologous to the small inverted repeat transposable element (MITE). With the proviso that the first primer is capable of hybridizing to a small inverted repeatable transposable element (MITE) in the DNA, and the second primer is or is not identical to the first primer, and D) separating the desired extension products by size; e) the pattern of the extension products It consists of reconstructing the genomic from and f) overlapping patterns; measured.

According to the present invention there is also provided a method for mapping a polymorphic genetic marker, comprising: a) providing a mixture of nucleic acid sequences digested with a restriction enzyme from a biological sample from a eukaryotic organism. B) combining the enzymatically digested nucleic acid sequence mixture with a first inverted homologous repetitive transposable element (MITE) primer, a fragment or derivative thereof, and a second primer.
Amplify using primers, except that the first primer is specific for MITE and the second primer is identical or not identical to the first primer;
And c) identifying a set of separately amplified nucleic acid sequences in the mixture; and d) identifying at least one of the separately amplified nucleic acid sequences as homologous or non-homologous to the MITE sequence; Mapping to the genetic polymorphism, thereby providing a marker for the polymorphism.

The marker system of the present invention based on MIT differs from any of the prior art approaches and is simpler, gives more information and is repeatable.

For the purposes of the present invention, the following terms are defined as follows: The term "MITE" is intended to mean a small inverted repeating transposable element. In fact, MITs are a superfamily of transposable elements. These elements are less than 3 kilobases in length, contain complete or degenerate terminal inverted repeats, have a target site overlap of less than or equal to 10 base pairs, and Medium to very rich.

MITes are less than 1 kilobase in length, contain complete or degenerate inverted repeats, have TA or TAA target site duplications around, and are moderate to very abundant in the genome .

The term “MITE-based primer” is intended to include primers comprising MITE or a fragment thereof, as well as primers derived from MITE and primers which recognize and hybridize or anneal to MITE.

The term “MITE-based genetic marker” (MGM) is a marker that hybridizes to a MIT element, or at least one MIT primer and optionally another ITE primer or SSR sequence, retro element sequence, RELP sequence or gene sequence. Is intended to mean a marker produced by PCR amplification of a nucleic acid sequence.

The term “inter-MITE polymorphism” (IMP) relates to a subset of MGM and means a marker obtained by PCR amplification of a nucleic acid sequence using one MITE primer or two different MITE primers Intended.

The term “eukaryote” or “eukaryotic organism” is intended to mean plants, animals and fungi. The term homologous is intended to mean a nucleic acid sequence that hybridizes under stringent conditions to the complement of the homologous nucleic acid sequence in the context of the homologous nucleic acid sequence.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides the novel gene markers referred to herein as MITE-based gene markers (MGMs). In this novel method using PCR, polymorphisms are represented using primers designed from abundant transposable elements, MITE. The utility of these transposable element-based primers has been demonstrated in barley-doubled haploid mapping groups and in barley (Hordeum vulg).
are varieties) and wild barley (Hordeum spontaneu)
m) One system was determined by studying segregation patterns in genotyping. In accordance with the present invention, a novel type of DNA markers, referred to herein as MITE-based genetic markers, and the chromosomal localization of these markers, their universality and versatility, and finger Provides printing results. Finally, applicant considers the feasibility and generalization of the MGM and IMP approaches of the present invention.

Advantages and Improvements Over Existing Technology As noted above, MITE members are often found in association with genes, ie, are not restricted to repeat regions. The widespread use of MIT has great value. It shows that virtually any region of the genome is susceptible to IMP amplification in many eukaryotic organisms.

A total of 50-100 scorable bands were amplified with any single primer and M
This indicates that ITE is present in the genome at a high copy number. With several primers and 50-100 loci per primer, the entire genome can be quickly covered in the screen.

MITE primers can be combined with other types of primers, such as primers specific for SSR, retroelement, sequenced RFLP, random genomic sequence, vector sequence, and gene. This certainly increases the potential of the MGM method of the present invention.

The method of the present invention, in combination with a high-resolution LI-COR automated fluorescent genotyping system, provides tremendous power to DNA mapping and fingerprinting techniques. Its power and resolution over RAPD and RFLP are evident as more loci can be detected in a single reaction. MGM and IMP analysis is easy, fast, and cost-effective. In contrast to RAPD analysis, significantly fewer primers are required. Unlike AFLP and RFLP technologies, MGM and IMP do not require restriction enzyme digestion or adapter ligation.

Description of the Technology i) The mapping groups that used plant material were the barley variety Lina and the barley wild variety Cana.
It consists of 88 doubled haploid individuals from crosses with da Park. This group has been used mostly to construct linkage maps based on RFLP markers.

[0036] 26 barley entries and one barley wild species entry, Canad
A total of 27 varieties, including a Park (see Table 1), were used for fingerprinting and used with Lina as parent to generate mapping groups. Recoveries included double and six-row types. Within the double-row type, both spring and winter varieties were included. All of the 27 varieties were previously used for RFLP genotyping studies, and thus, RLFP-based genetic correlations between these varieties were known.

[0037]

[Table 1]

Ii) PCR Detection System Two detection systems were used to compare the resolution and effectiveness in identifying polymorphisms. The first was a conventional agarose detection system. In this system, PCR was performed using normal primers (unlabeled). The PCR product was Nu
Using sieve agarose (2/3 Nusieve: 1/3 agarose),
Or visualized on a 2% agarose gel without use. Second was the LI-COR automated DNA sequencing / genotyping system. Primers for this system were IRD700 ™ fluorescent dyes (Li-COR, Inc.,
Licoln, Nebraska). PCR products were visualized on a 6% acrylamide denaturing gel using a 41 cm long glass plate apparatus. Gel electrophoresis was performed on a LI-COR 4200 system.

Iii) PCR Seven master primers (Table 2) and their 3′-anchor derivatives (Table 3)
) Was designed and evaluated in this study. The six master primers are ITE primers (TEM-1, TEM-2, TEM-3, TEM-10, TEM-11).
And TEM-12), and TEM-4 has several Tyl / copia-like retrotransposons (Hirochika H. and R. Hiroc).
hika 1993. Tyl-copia group retrotran
sponsons as ubiquitous components of
plant genomes. Jpn. J. Genet. 68:35
-46. ) Was part of the conserved sequence of the reverse transcriptase (RT) domain. The master primer was denatured if one or more nucleotides could be in one position. The anchor primer was the master primer with additional nucleotides added to the 3 'end of the master primer (Table 3). MITE primers were designed from consensus sequences in the terminal inverted repeat (TIR) region of MITE from each category. Primers were designed using both TIRs. T
Only EM-4 was used in combination with other primers. Primers were used in the agarose gel detection system and the LI-COR automated detection system, except that the primers for the latter were not labeled with fluorescent dyes.

[0040]

[Table 2]

[0041]

[Table 3]

The PCR amplification for the agarose detection system was 2.5 mM MgCl ₂ , 0.4
mM dNTPs, 1 μM of each primer and 0.625 units of AmpliT
25 μl containing aq ™ DNA polymerase (Perkin-Elmer)
Performed in volume. The following profiles were used: initial denaturation at 94 ° C for 1 minute 30 seconds,
35 cycles of 94 ° C for 30 seconds, 58 ° C for 45 seconds and 72 ° C for 1 minute, and 7
Final extension at 2 ° C. for 5 minutes. An annealing temperature of 60 ° C. was used whenever including TEM-1.

The PCR amplification for the LI-COR detection system was performed under the same conditions as in a normal agarose system, but using a total reaction volume of 20 μl and 0.5 units of AmpliTaq DNA polymerase (Perkin-Elmer). Except what was done. Similar general profile (without TEM-1 temperature change)
It was used. PCR amplification was performed in two steps. The first step is pre-amplification,
Unlabeled primers were used for 35 cycles. A 3 μl aliquot of the pre-amplification mix was used for the second step of amplification. A 0.1 μM concentration of labeled primer was used for the second round of amplification (compared to 1 μM unlabeled primer in the first step).

Iv) Data collection and statistical analysis a) Fingerprinting and gene similarity analysis Polymorphisms and common bands are present (1), absent (0
) Or data not collected (9). The resulting raw data matrices are Nei and Li (Nei M. and W. Li 1979. M
athletic models for studying gene
tic variation in terms of restriction
n endoncleases. Proc. Natl. Acad. S
ci. 76: 5269-5273) _{methods, 2n xy / (n x +} n y) ( where, n _x and n _y are each the number of bands in lines x and y, n band _xy is shared by both lines The relative gene similarity (GS
) Used to generate the matrix. GS values are calculated using both the polymorphism and the common band.

The phylogenetic tree is based on the GS matrix and is based on UPGMA (unweighted).
pair-group method arithmetic average
). A combined phylogenetic tree resulting from analysis using two MITE primers was created. Standardized Mantel statistics (Mantel
N. A. 1967. The detection of disease
clustering and a generalized regress
ion approach. Cancer Res. 27: 209-22
0) was used to compare genetic similarity matrices based on MIT-based genetic markers with the same breed genetic similarity matrix based on 313 polymorphic RFLP marker bands. Testing for significance was performed by comparing the observed Z values with the distribution of 1000 random permutations of the matrix. All statistical analyzes were performed on NTSYS-pcsoftware (Rohlf FJ. 1).
994. NTSYS-pc numerical taxonomy and
multivariate analysis system, versi
on 1.80, Exeter Software, N.M. Y. ).

B) Gene Mapping Localization of a MIT-based gene marker prepared using TEM-1 and TEM-10 primers was performed, and a barley cultivar Lina population × H. s
These were mapped within the framework of the 71 RFLP markers used to generate a map of the pontanum Canada Park population. A subpopulation of 88 doubled haploids of this population was used for mapping. The segregation was analyzed using the chi ² analysis. Mapping is performed by the computer program MAPPACKER (La
nder E. S. , P .; Green, J.M. Abrahamson, A .; Ba
rlow, M .; J. Daly, S.M. E. FIG. Lincoln and L.A. Newburg
1987, MAPMAKER: An interactive compound
er package for constructing primary geo
netic link map of experimental and
natural populations. Genomics Volume 1: 174-1
81). A MIT-based genetic marker with a threshold of 2
Linked groups were identified using a two-point analysis at LOD threshold 4, excluding the 7H group forming the group, and associated based on published RFLP marker localization. Markers were placed within linkage groups using multipoint analysis at LOD threshold 2.

Results To evaluate the MITE sequence in the PCR-based method of the present invention, primers were designed from the terminal inverted repeat (TIR) region, and all primers were designed outward from the TIR. In this way, it is expected that any sequence amplified by these primers will be located between two adjacent MITTES within an amplifiable distance. These primers are used for barley cultivar Lin
a and H. It is used alone or in combination in the separation analysis using a doubled haploid population of 88 individuals obtained from the cross of the Spontanum cultivar Canada Park.

A) Single Primer Amplification Each MITE primer generated about 10 enumerable bands on an agarose gel, of which 2-5 were polymorphic. These polymorphisms indicate that the barley parent strain L
ina and H .; It was clearly detected among the spontaneum parent strains Canada Park, and many showed the expected 1: 1 Mendelian segregation in doubled haploids. FIG. 1 shows an example of the separation pattern.

M indicates the λPstI marker. Lane 1 contains the PCR product of barley Lina. Lane 2 is H.E. Includes PCR product of Spontaneum Canada Park. Lanes 3-28 contain the individual PCR products of the mapped population.

The primer TEM-1 showed a very weak and high background with almost invisible bands. Presumably this is due to a number of closely related sequences, such as those resulting from mutations in the TIR region. In this case, formaldehyde has been reported to reduce PCR background and increase specificity (Nagaoka T. and Y. Ogihara 1997, A.
pplicity of inter-simple sequence
repeat polymorphisms in what and theei
r use as DNA markers in comparison to RF
LP and RAPD markers. Theor. Appl. Genet.
94: 597-602), 2% formaldehyde was added to the reaction mixture.

A total of about 100 counted bands were ligated using the primer TEM-1 to LI-
Detected on COR sequencing gel. 6 when primer TEM-10 is used
0-70 lines could be detected and 30-40 lines could be detected with primer TEM-3. FIG. 2 shows a fragment of acrylamide gel electrophoresis using TEM-1. As with the agarose detection system, the barley parent strains Lina and H. spontan
The polymorphism was clearly detected among the parent strains of Canada, Parka Park.
It showed a 1: 1 Mendelian segregation in the doubled haploid population.

Lane 1 contains the PCR product of the barley parent strain Lina. Lane 2 is H.E. spo
Includes PCR product of ntaneum parent strain Canada Park. Lanes 3-4
5 is the individual PC of the population obtained from the cross of Lina × Canada Park
Includes R product.

B) Primer Combination A primer combination test was performed simply using an agarose detection system. Several situations were encountered when using these primers in different combinations. TEM-
The 4 / TEM-10 combination produced a similar pattern as TEM-10 alone, whereas the TEM-1 / TEM-10 combination produced different results. That is, TE
Many of the bands obtained with M-10 alone were inhibited, and the bands obtained with TEM-1 alone were barely visible, with smaller sized bands appearing. TEM-3 /
Increasing the extension time with the TEM-10 combination (1 min 15 sec) produced a different pattern with three distinctly visible separation bands different from either primer alone (FIG. 3). Lanes 1 to 16 are individual individuals in the individual population mapped. Parent stock is not shown.

A similar situation was seen when using primer TEM-1. The band pattern did not change when TEM-1 was combined with TEM-4 (FIG. 4).
A), the pattern changed when this primer was combined with TEM-3 or TEM-10. In addition, if the extension time is extended (1 minute 15 seconds), TEM-1 / TE
The combination of M-4 produced a longer separation band, and some other bands were suppressed (FIG. 4B). Interestingly, the longer band (T
1-4AA) separated in much the same way as band T4-10A (FIG. 1).
. T1-4AA and T4-10A show bands T4-10A even with TEM-10 alone.
Are not products of the same common primer TEM-4. Also, T1-
4AA is about 300 bp longer than T4-10A. This indicates that the combination of the two primers amplified the tightly linked region of DNA. This is not surprising as these exchangeable sequences are expected to be present at high copy numbers.

The description of FIGS. 4A and 4B is the same as that of FIG. Lane numbers are shown in FIGS. 4A and 4B.
Correspond to each other. Some primer combinations showed inconsistent results. Possible explanations are as follows: Each primer alone is amplifying more than 10 bands, and therefore the combination of these primers may not be clearly visible due to too many bands generated or only minor changes in state Different annealing temperatures (as with TEM-4 with very low annealing temperatures) may be an important factor in determining the generated pattern. And the affinity of the different primers may result in the predominance of certain bands, such as with TEM-1 and TEM-10.

Nevertheless, using a fluorescently labeled detection system, as in the case of a single primer reaction, some of these problems are solved, and the combination of primers increases the number of detectable positions. Seem.

C) Chromosomal Localization of Gene Markers Based on MITE Using an agarose detection system, three types of MITE primers and Ty1 / copi
a Retrotransposon primer generated a total of 15 detectable polymorphic markers on the mapped population. All but two were separated at the expected 1: 1 separation ratio. Thirteen of these markers will be placed on the map.
The other two markers remain unrelated. These are markers that show a significant deviation from the expected separation ratio and appear to consist of two bands of similar size that cannot be separated on agarose.

In FIG. 5, MIT-based genomic markers are seen in larger font (Degesh Japan font) and in bold. Only loci detected on acrylamide gels using fluorescently labeled TEM-1 and TEM-10 are visible. Loci in parentheses are those that could not be evaluated with an LOD score of 2 or higher. Approximately 120 and 90 distinct bands are shown in the LI-COR sequencing gel using primers TEM-1 and TEM-10, respectively.
l) detected above. The range of the size of the detected band is approximately 100 bp to
It was 1 kb. Visualized by polyacrylamide gel electrophoresis (vi
FIG. 2 shows a part of the results of amplification using TEM-1 in the same manner as described above.

[0059] 75 polymorphic bands and 19 polymorphic bands were generated using TEM-1 and TEM-10 primers, respectively. Some pairs of bands show co-dominant behavior.
tbevior (locus T1-0.2 on 1H, loci T1-4 and T1-16 on 2H, loci T10-6 on 3H, loci T10-8 and 7H on 4H. Upper locus T1-36; FIG. 5). However, the remaining bands are exactly 41 from the Lina parent and exactly H. s
A presence / absence pattern with 41 from the pontanum progenitor was shown.
Of the 70 mapped TEM-1 loci, 24 loci significantly deviated from the expected 1: 1 segregation ratio. All 24 loci except one (T1-19 on 7H) were RFLP
Markers were also located in regions that showed distorted separation rates in this mapping population. Two out of the 18 TEM-10 loci deviated from the expected segregation rate. These were again confirmed in regions where the RFLP locus also deviated from the expected 1: 1 segregation ratio.

[0060] A total of 88 loci were mapped. These loci covered all seven linkage groups (FIG. 5). Furthermore,
The distribution of these loci depends on the region of the centromere where recombination is typically induced (
It showed no significant accumulation except for the expected clustering around the centromeric regions (eg, the 1H, 3H and 7H groups, FIG. 5). In fact, this distribution is based on the cDNA detection RFLP (cD
Same as the distribution found for NAs detecting RFLPs) (LSO'Dongough, unpublished). This suggests that MITE is located in the region of the genome-containing coding sequence, and that it is possible to cover this entire genome with a limited set of MITE-based primers.

D) Fingerprinting vulgare species Lina, H .; spontaneum species Canada Park (Canada Park) and 25H. vulgare cultivar (cul)
A total of 27 cultivars, including var. vars, were used to evaluate the utility of these MITE primers in fingerprinting. On agarose, these primers and primer combinations
TEM-3, TEM-10, TEM-1 / -3 and TEM-1 / -4 were found to be useful in identifying these cultivars. One to three polymorphic bands were seen for each of these primers and combinations. An example of a fingerprinting experiment on agarose is shown in FIG.

The three separation bands in FIG. 6 indicated that 27 cultivars were divided into seven groups. M represents the molecular weight marker λPstI. The number corresponds to the number in Table 1.

The two MIT primers, TEM-1 and TEM-10, were combined with a fluorescent labeling detection system.
on system) in fingerprinting analysis.
For TEM-1, a total of 62 bands were obtained, of which 37 were polymorphic and the remaining 22 were the same as all 27 cultivars. This electrophoresis 1
The section is shown in FIG. For TEM-10, a total of 60 bands were obtained, of which 34 were polymorphic and the remaining 26 were the same as all 27 cultivars. Confirmation of the line of FIG. 7 (identification)
Can be found in Table 1.

Lanes 1 to 27 are Lina and Canada Par, respectively.
k, Alexis, Angora, Ariel, Azul, Ellice, E
xpress, Filippa, Goldie, Golf, High amylose glacier, Igri, Ingri
d, Kinnan, Maud, Meltan, Mentor, Mette, Mo
na, Roland, Saxo, Svani, Tellus, Tofta, Tr
Figure 7 shows results from ebon and Vixen. Dashes indicate markers that distinguish at least one cultivar from other cultivars.

Using the coefficients of Nei and Li (Nei and Li, supra), the TEM
-1, TEM-10 and both primer binding data (combined
A GS matrix was obtained for data). A dendrogram was obtained for the same data combination. TEM-1 and T
FIG. 8 shows a dendrogram of the binding data of EM-10. This dendrogram is described in The Spontaneum strain is H. vulgare cultivars. There is an exception of Azul (six-row type), but spring two-row types are clustered, and four winter types (A) are included in the present invention.
ngora, Express, Igri and Vixen). High Amylose Glacier (High Amy)
(Lose Glacier) line is a 6-row type. As a result of comparing the GS matrix with the GS matrix previously obtained for the RFLP analysis,
The el statistic was Z = 0.69475, indicating a good correlation between the two.
This positive correlation is very significant with the probability P = 0.0020, and it is believed that this value of Z would only be obtained by chance.

E) Universality of the primers In order to confirm the universality of the primers, animal-derived and plant-derived ITE master primers were used for plant genomic DNA, insect genomic DNA and human genomic DNA. .

FIGS. 9A, 9B, 9C and 9D are as described in Table 2. Master primer TEM-12 (FIG. 9A); master primer TEM-1 (FIG. 9B);
Master primer TEM-10 (FIG. 9C) and master primer TEM-
11 (FIG. 9D) shows typical results of PCR-amplified profiles of seven different DNA sources. Sources of DNA (listed above for each lane) are as follows: 1) normal human DNA (male) 2) normal human DNA (female) 3) human DNA (male with albinism) 4) Human DNA (female with albinism) 5) Insect: Trichogamma 6) Legume: Soy 7) Legume: Alfalfa 8) Brassicaceae: Canola 9) Cereals : Wheat 10) Cereals: oat 11) Cereals: barley These results clearly demonstrate that MITE-based markers can be used in a wide variety of species.

F) Versatility of the Master-Derived Sequence To support the versatility of the MITE-based marker system, the master primer sequence is added by adding additional nucleotides to the 3 ′ end of the master primer sequence. Qualified. This has the effect of increasing the specificity of the amplified product, and it can be seen that the amplification profile obtained by the master sequence is too complex for TEM-1 primers derived from Stowaway. Especially useful when difficult.

FIGS. 10A, 10B, 10C, 10D and 10E show the preamplification stage (pream
FIG. 4 shows an example of the results obtained for the master primer TEM-1 in a replication step and its corresponding anchored primers listed in Table 3 for the amplification. These figures show the profile of cereal DNA (barley) with only the master primer TEM-1 (FIG. 10A);
(FIG. 10B); anchor primer TEM-1C (tied with "C") (FIG. 10C); anchor primer TEM-1G (tied with "G") (FIG. 10D) and anchor primer TEM-1T ("T"). FIG. 10E shows the profile amplified by the polymerase chain reaction (PCR) of cereal DNA (barley) as compared to that of FIG. 10E.

These results show that a simpler amplification pattern is obtained when TEM-1 is anchored at its 3 ′ end with any of A, C, T or G. Is evident. It is also clear that different complementary amplification patterns can be obtained using different 3 'terminal anchors.

General Purpose and Commercial Applications Various studies have shown that transposable elements are present in virtually all species studied to date. Retrotransposons are present at high copy numbers in the plant genome. The Alu family has 5 × 10 ⁵ per haploid human genome.
It is estimated to be a copy, in other words, there is one Alu factor for every 5 kb of DNA. This factor alone accounts for 5% of the genome in primates (Berg DE and MM Howe, 1989. Mobile DNA.
Washington, American Society of Micr
obiology). Ty1 / copia group factor is wide diversity observed between species, it accumulates to genome per ¹⁰⁶ copies in Vicia species (Vicia species), may constitute more than 2% of the genome (Pearce S.R.
, H .; Gill, D.A. Li, J. et al. S. Heslop-Harrison,
A. Kumar and A.M. J. Flavel 1996. Ty1-copier group retrotransposons in Vicia faba species: copy number, sequence heterogeneity (seq
ence heterogeneity) and chromosome arrangement. Mol. Gen.
Genet. 250: 305-315). The BARE-1 retrotransposon has a copy number of 3 × 10 ⁴ and 6.
Up to 7% (Suoniemi A., K. Annamawat-
Jonsson, T .; Arna and A. H. Schulman 1996
. Retrotransposon BARE-1 is a (Yabane) barley (Hordeu)
m vulgare L. ) The main distributed component of the genome. Plan
t Molecular Biology 30: 1321-1329). Sa
A study by nMiguel et al. (SanMiguel P., A. Tikh
onov, Y .; -K. Jin, N.M. Motcholskaia, D.M.
Zakharov, A .; Melake-Berhan, P .; S. Sprin
ger, K .; J. Edwards, M.E. Lee, Z .; Avramova and J.M. L. Bennetzen 1996. Nested retrotransposons in the intergenic region of the maize genome. Science 274: 765-7
68), Maize Adh isolated on a yeast artificial chromosome (YAC) clone
Sequencing of the flanking 280 kb region flanking the 1-F gene revealed 37 classes of small transposon repeats, accounting for more than 60% of the clones.

Factors of Tourist and Stowaway (Bureau TE and S
. R. Wessler 1992. Tourist: A large family of small inverted repeat factors, often associated with maize genes. Plant Ce
11: 1283-1294; and Bureau T. et al. E. FIG. And S.I. R.
Wessler 1994. Stowaway: A new family of inverted repeat factors associated with both monocotyledonous and dicotyledonous genes. Plant
Cell 6: 907-916) are traditional TIRs such as Ac and En / Spm.
Although significantly different from the transposable element family, they are members of the TIR class of transposable elements. Barfly, a new member of the TIR transposable elements such as Tourist and Stowaway, has been found to be associated with the (yabane) barley xylose isomerase gene. These factors, collectively referred to as MITes, along with several other factors of this type (Bureau TE, PC
. Ronald and S.M. R. Wessler 1996. Computer-based systematic surveys have revealed the predominance of small inverted repeat factors in wild-type rice genes. Proc. Natl. Acad. Sci. 93: 8524
-8529) has been found in a number of plant species studied so far. M
ITEs are also expected to be present at high copy numbers in eukaryotic genomes.

The ubiquity and distribution of transposable elements in the genome suggests that they can be used as PCR-based mapping tools. In fact, Sinnet
(Sinnet D., J.-M. Deragon, L.R.
d. Labuda 1990. Alumorphs-human DNA polymorphism detected by replication chain reaction using Alu-specific primers. Genom
ics 7: 331-334) disclose Alu-specific primers with different human DNs.
It was used to search for polymorphisms between A samples. These investigators have clearly shown the feasibility of using these polymorphisms (referred to as alumophths) as a tool for genomic analysis (Sinnet et al., Supra), and using these almoss Succeeded in detecting the linkage of a single Almov to a human disease (
Zietkiewicz E. , M .; Labuda, D.A. Sinnet,
F. H. Glorieux and D.E. Labuda 1992. Linkage mapping by simultaneous screening of multiple polymorphic loci using Alu oligonucleotide-directed PCR. Proc. Natl. Acad. Sci. 89:84
48-8451). The copier-like retrotransposon, PDR1, is also useful for studying polymorphisms and, together with other specific primers, Pisum
Has been used successfully to diagnose different systems in (Lee D., T .;
H. N. Ellis, L .; Turner, R.A. P. Hellens and W.C.
G. FIG. Clear 1990. Copier-like factors in Pisum demonstrate the use of dispersed repetitive sequences in genetic analysis. Pla
nt Molecular Biology 15: 707-722).

In the present invention, the TIR transposable element members, MITEs, are used as mapping and fingerprinting tools in (Yabane) barley, and are used in both conventional agarose and LI-COR automated DNA analysis systems. . We successfully detected polymorphisms within the vulgare species group, mapped these MGMs to existing genetic linkage maps, and created fingerprints of (cultivated) varieties.

In a conventional agarose detection system, we detected 15 clearly evaluable polymorphisms using three MITE primers and one retrotransposon primer and 13 out of 15 Were mapped to four linkage groups of (Yabae) barley. Each MITE primer or primer group produced more than 10 scorable bands, with 2-5 being polymorphic. In the IL-COR automated genotyping system, each of the two MITE primers shown resulted in nearly 100 scorable bands, with up to 75 being polymorphic. Markers for mapping all seven (Yabane) barley linkage groups were obtained using these two primers. This indicates a random distribution of MIT-based markers in the genome.

New ITEs are constantly being discovered by computer-based sequence similarity searches. As the number of MITes increases, virtually any type of detailed linkage map with a high copy number of MITes can be easily constructed based solely on MGMs. Linkage studies of important genes with MGMs can also be easily performed. The high level of diversity detected by these MITE-based primers among (cultivated) varieties within the same species demonstrates the practical value of these primers.

Some replaceable components, such as Alu (Berg and Howe, supra; Makalo
wski WGA Michell and D. Labuda 1994.Alu sequences in the coding r
egions of mRNA: a source of protein variability. Trends Genet. 10: 188
-193), and the mouse B2 component (Clemens MJ 1987. A potential role fo
r RNA transcribed from B2 repeats in the regulation of mRNA stability.
Cell 49: 157-158) was often found to be associated with the gene. M
ITE constituents are also often identified in plant and other eukaryotic genes. Stowaway was first identified as a cause of mutation at the maize wx locus (Bureau and Wessler, supra). More than 100 genes have been found to carry MITE in their coding and non-coding regions (Bureau et al.
al., supra). Multiple gene sequences or characteristics of gene sequences due to the close association of retro components with animal and plant genes, and the close association of MITE with genes in agronomic crops and other plants A new way to make decisions has been opened. Indeed, research has focused on the isolation of gene sequences (
Nelson DL, SA Ledbetter, L. Corbo, MF Victoria, R. Ramirez-Soli
s, TD Webster, DH Ledbetter and CT Caskey 1989.Alu polymerase
chain reaction: A method for rapid isolation of human-specific DNA seq
uences from complex DNA sources.Proc. Natl. Acad. Sci. 86: 6686-6690
; Souer E., F. Quattrocchio, N. de Vetten, J. Mol and R. Koes 1995. A g
eneral method to isolate genes tagged by a high copy number transposable
element. Plant Journal 7: 677-685) in genome analysis (Hirochika H.
1997. Retrotransposons of rice: their regulation and use for genome a
nalysis.Plant molecular Biology 35: 231-240; Lee D., THN Ellis,
L. Turner, RP Hellens and WG Cleary 1990. A copia-like element i
n Pisum demonstrates the uses of dispersed repeated sequences in genetic
plant Molecular Biology 15: 707-722), and in the analysis of gene structure and gene expression (White SE, LF Habera and SR We
ssler 1994.Retrotransposons in the flanking regions of normal plant ge
nes: A role for copia-like elements in the evolution of gene structure
Natl. Acad. Sci. 91: 11792-11796), using other types of replaceable components.

There are several forms of application of the method of the invention. 1) Linkage studies a) Linkage maps can be constructed using MITE markers as described herein. This requires that the population and parent be separated. Linkage maps are constructed based on separation.

B) Linkage with phenotypic traits or genes can also be performed. This was accomplished by combining volume-identified segregant analysis to facilitate research. In this case, the two parental and phenotypically (with trait) or genetically (with gene) pools are used in PCR amplification with MITE primers to identify polymorphic markers, And thus the putative linkage can be identified.

C) According to the same principle, the relationship between MGM or IMP and quantitative trait loci (QTL) that regulates traits under complex gene regulation is determined by single point ANOVAs (single point ANOVAs). , Interval mapping (Interval Mappi
ng) and various statistical analyzes such as mixed interval mapping.

D) Once the linkage of markers with agronomically important traits is known, these markers can be used in marker assisted selection (MAS) to facilitate breeding programs . 2) Fingerprint studies MGM and IMP approaches are used to help identify cultivars, helping to construct large insert libraries, such as YACs (yeast artificial chromosomes) and BACs (bacterial artificial chromosomes). And can assist in marker conversion as well as gene isolation.

A) The generated MGM and IMP markers can serve as landmarks in aligning contigs and during chromosome walking. b) The MGM and IMP approaches determine their pedigree and genetic relevance in fingerprinting cultivars and in breeding lines, and determine the degree of contribution of parent lines to offspring lines. , And can be easily explored in performing new line and cultivar assays.

The MGM approach can be used to assist in gene isolation and subcloning of genomic sequences. a) If a gene is tagged with a replaceable component, the MGM can be used because it is entirely present in the genome. MITE primers can be used in combination with primers designed from transposons for tagging.
Flanking sequences can be amplified and subsequently used to isolate wild-type genes. Compared to conventional cloning of transposon-tagged genes, this approach saves one round of DNA library screening.

B) Using a similar scenario for gene isolation, MGM can be used to isolate genomic sequences flanking known gene sequences. MITE primers and primers designed from known gene sequences can be used in PCR to amplify adjacent sequences.

C) RFLP markers can be converted to PCR-based markers using amplification using primers from DNA clones that detect RFLP used in combination with MITE primers.

Although the invention has been described in conjunction with specific embodiments of the invention, it will be understood that further modifications are possible and that this application will generally be in accordance with the principles of the invention. And, as would be within the scope of known or customary practice in the art to which this invention encompasses, and as applicable to the essential features described above, such It will be understood that it is intended to cover any variations, uses, or adaptations of the invention, including new developments, and in accordance with the scope of the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows primer combination TEM-4 / -10 on agarose gel.
Alternatively, a PCR product of TEM-10 alone is shown.

FIG. 2 shows a section of the results of a PCR of IRD700 ™ fluorescent dye-labeled TEM-1 primers, using a LI-COR automation system 4200 and a 6% acrylamide gel according to a preferred embodiment of the present invention. Above, where P1 is the parent H. vulgare, and Lina (P1) and P2 are parent H. vulgare. spontane
um, Canada Park (P2), and the separated individuals were Lin
a and from the cross between the Canada Park DH (double haploid) population.

FIG. 3 shows TE on agarose gel with longer extension times of 1 minute and 15 seconds.
The result of PCR of M-3 / -10 is shown.

FIG. 4 shows T showing different products with an extension time of 60 seconds and an extension time of 75 seconds.
FIG. 4 shows the results of PCR on agarose gels of EM-1 / -4.

FIG. 5 shows IMP detected using TEM-1 and TEM-10 primers.
Showing the distribution of loci; vulgare cv. Lina x H .; sponta
Figure 2 shows a linkage map of the neucanada Park population.

FIG. 6 shows the fingerprinting of 27 Hordeum lines on agarose gel.

FIG. 7 shows a section of the results of fingerprinting of 27 hordeum systems using IRD700 ™ fluorescent dye-labeled TEM-1 primers.

FIG. 8 is a graph showing two bands based on the band formation patterns of TEM-1 and TEM-10.
7 shows dendrograms resulting from UPGMA clustering of the gene similarity matrices of seven varieties.

FIGS. 9A, 9B, 9C and 9D show master primer TEM-12 (FIG. 9A).
); Master Primer TEM-1 (FIG. 9B); Master Primer TEM-1
0 (FIG. 9C) and master primer TEM-11 (FIG. 9D). "
M in different eukaryotes showing PCR amplification profiles of 11 different DNA sources
2 illustrates the universal use of ITE based markers.

FIGS. 10A, 10B, 10C, 10D and 10E show master primers (T
An example of the results obtained using EM-1) and its corresponding anchoring primer is shown.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZW (72) Inventor Bureau, Thomas Quebec, H. 2 Double 2E7, Montreal, De Brion 4250 (72) Inventor Chan, Ruin 38104, Tennessee, United States of America 38, Memphis, Central Avenue 2639, A Part buoy-4 (72) Inventor Randli, Benoir Quebec, Canada Jay 2 W 1 Bee 3, Lacadie, Are de Cigal 134 (72) Inventor Odnougue, Louise Stefa Knee Canada Quebec Jay 3H 2Es 5, Mont Saint-Hilaire, Doral-Desormo 524 F-term (reference) 4B024 AA11 AA20 CA03 CA09 HA14 4B063 QA12 QQ07 QQ09 QQ42 QR32 QR55 QR62 QS12 QS25 QX02

Claims (20)

[Claims] 1. A method for detecting a polymorphism in a nucleic acid sequence of interest, comprising the steps of: a) a first primer homologous to a small inverted repeatable transposable element (MITE). , A fragment or derivative thereof, and a second primer that anneals to the MITE when the first primer is present in the nucleic acid sequence of interest.
Amplifies with a primer, except that the second primer is identical or not identical to the first primer and is or is not homologous to the MITE sequence; b) the interest of interest amplified in step a) Isolating a fragment of the above nucleic acid sequence; and c) obtained by amplifying a nuclear sequence using at least one primer to detect a difference in nucleic acid sequence between the fragment obtained in step b) and a reference. Analyzing a fragment obtained in step b) with respect to a reference fragment, whereby the difference is indicative of a polymorphism in the nucleic acid of interest. 2. A method for genotyping a eukaryote, comprising the steps of: a) replacing the nucleic acid sequence of interest with a first primer homologous to a small inverted repeat transposable element (MITE) fragment or Amplifies with its derivative, and a second primer, provided that the first primer anneals to the MITE when present in the eukaryotic nucleic acid sequence and the second primer is identical to the first primer. Or not homologous or not homologous to the MITE sequence; b) isolating the fragment of said nucleic acid sequence of interest amplified in step a); and c) obtaining in step b) Comparing the fragment obtained with the reference nucleic acid sequence from the eukaryote, whereby the identity of the fragment and the reference nucleic acid sequence in step b) is indicative of the eukaryote having the nucleic acid sequence. The above method comprising: 3. A method for fingerprinting a eukaryotic organism, comprising the steps of: a) combining a nucleic acid sequence of the eukaryotic organism with a first primer, a fragment or derivative thereof, and a second primer homologous to MITE. Amplifies, with the proviso that the first primer is specific for the MITE sequence and the second primer is identical or not identical to the first primer and is homologous or not homologous to the MITE sequence. B) separating a fragment obtained from amplifying the nucleic acid sequence of step a), whereby the fragment thus isolated is typical of a eukaryotic organism. 4. The method according to claim 1, wherein the step of amplifying is effected by a PCR technique. 5. The method according to claim 1, wherein the first primer is Tourist, Storey Way, Bar.
3. Derived from a consensus sequence from fly or Mariner.
5. The method according to 3 or 4. 6. The first primer comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 consist of 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35 The method according to claim 1, 2, 3, 4, or 5 having a nucleic acid sequence selected from the group. 7. The second primer is a MITE sequence-specific primer, a SSR sequence-based primer, a retro element sequence-based primer RELP-directed cloned nucleic acid sequence-based primer, a random genomic sequence-based primer and a gene. Selected from the group consisting of sequence-based primers,
The method of claim 1, 2, 3, 4, 5, or 6. 8. The method of claim 1,2,3,4, for tracking progeny of a eukaryotic organism.
Use of a polymorphism detected by the method of 5, 6 or 7. 9. The method of claim 1, 2, 3 for measuring hybridization in a eukaryotic organism.
Use of a polymorphism detected by the method of 4, 5, 6, or 7. 10. A method for identifying variations in a linked phenotypic trait.
Use of a polymorphism detected by the method according to claim 1, 2, 3, 4, 5, 6 or 7. 11. Use of a polymorphism detected by the method according to claim 1, 2, 3, 4, 5, 6, or 7 as a genetic marker for constructing a genetic map. 12. The method of claim 1, 2, 3, 4, 5, or 5 for identifying individual progeny from a cross, said progeny having a desired gene contribution from a parent donor and / or recipient parent. Use of a polymorphism detected by the method of 6 or 7. 13. The method of claim 1, 2, 3, 4, 5, 6, or 7 for isolating a genomic DNA sequence surrounding a gene coding or non-coding DNA sequence.
The described method. 14. The method according to claim 13, wherein the genomic DNA sequence surrounding the gene coding DNA sequence is a promoter sequence or a control sequence. 15. A nucleic acid fragment or derivative thereof for use as a probe on a nucleic acid sequence, obtained by amplifying a eukaryotic nucleic acid sequence with at least one primer homologous to MITE. 16. A nucleic acid fragment or derivative thereof according to claim 15, for marker-assisted selection (MAS), map-based cloning, hybrid assays, fingerprinting, genotyping, and allele-specific markers. Use of. 17. The method according to claim 2, wherein the eukaryote is a plant, animal or fungus. 18. The method of claim 3, wherein the eukaryotic organism is a plant. 19. A method of genomic mapping, comprising the steps of: a) fractionating a genomic of a eukaryotic organism; b) cloning the fractionated genomic into a vector; The vector so amplified is tested by amplifying the DNA in the cloned vector with a first primer and a second primer homologous to the small inverted repeat transposable element (MITE), With the proviso that the first primer is capable of hybridizing to a small inverted repeatable transposable element (MITE) in the DNA and the second primer is identical or not identical to the first primer and is homologous to the MIT sequence. D) separating the desired extension products by size; e) measuring the pattern of the extension products; It said method comprising the reconstitution of a genomic from to f) overlapping patterns. 20. A method for mapping a polymorphic genetic marker, comprising: a) providing a mixture of nucleic acid sequences digested with a restriction enzyme from a biological sample from a eukaryotic organism; b) the enzyme. The mixture of digested nucleic acid sequences is combined with a first inverted homologous inverted repeatable element (MITE) primer, a fragment or derivative thereof, and a second primer.
Amplify using primers, except that the first primer is specific for MITE and the second primer is identical or not identical to the first primer,
And c) identifying a set of separately amplified nucleic acid sequences in the mixture; and d) identifying at least one of the separately amplified nucleic acid sequences as homologous or non-homologous to the MITE sequence; Such a method, which comprises mapping to a genetic polymorphism, thereby providing a marker for the polymorphism.