CN1351671A - Transposon-based genetic marker - Google Patents

Abstract

The present invention relates to the use of DNA primers homologous to MITE in a method for detecting polymorphisms of a nucleic acid sequence. The method comprises the steps of amplifying nucleic acid sequences using a first primer homologous to a MITE in combination with a second primer homologous or not to a MITE, separating fragments of the nucleic acid sequences amplified, and analyzing the fragments obtained in relation to reference fragments obtained from amplification of a nucleic acid sequence with the primer homologous to MITE for determining polymorphism in the nucleic acid sequence. DNA primers homologous to MITE may also be used in genotyping, fingerprinting, mapping or cloning method in accordance with the invention.

Description

A novel genetic marker based on transposons

Background of the invention

(a) Field of invention

The present invention relates to a method for genotyping a nucleic acid sequence, the method uses a pair of primers to amplify, wherein the first primer has a homology with a miniature inverted repeat transposable element (miniature inverted-repeat transposable element, MITE) The DNA sequence of the second primer is the same as or different from the first primer. The present invention generally relates to the use of MITE primers for fingerprinting or linkage studies.

(b) Prior art

In McClintock (McClintock B. 1946. Genetics of Maize. Annals of Camegie Inst. Wash. 45: 176-186; and McClintock B. 1947. Cytogenetic studies of maize and Neurospora. Annals of Camegie Inst. Wash. 46: 146 -152.) Following the discovery of the transposable element Ac/Ds system, the genetic identification of a new transposable element system (family) became a plant (Peterson P. A. 1986. Mobile elements in maize. Plant Breeding Reviews 4 : 3-122.) and other hot areas in biogenetics research. This was followed by the molecular identification of transposable elements and the development of the use of these elements as tools for gene identification and isolation, particularly in the cloning of the white locus with the copia retrotransposon in Drosophila (Bingham P. M., R. . Lewis and G.M. Rubin 1981. Using a new general method to clone, the DNA sequence of the white site of Drosophila orangutan Drosophila. Cell 25:693-704.), and the maize transposable element Ac (Pohlman R.F., N.V. Fedoroff and J.Messing 1984. Nucleotide sequence of maize control element activator (Activator). Cell 37:635-643.) and En/Spm (Pereira A., Zs.Schwarz-Sommer, A.Gierl, I.Bertram, P.A. Peterson and H. Saedler 1985, Genetic and molecular analysis of the enhancer (En) transposable element system in maize (Zea mays). After molecular identification of EMBO J.4:17-25.). Since then, research on transposable elements has become a focus of biological science.

As in other areas of biological research, modern computer technology has accelerated the identification of transposable elements. Bureau et al. (Bureau T.E., P. C. Ronald, and S.R. Wessler 1996. A computer-based systematic survey revealed a large number of small inverted repeat elements in wild-type rice genes. Proc. Natl. Acad. Sci.93: 8524-8529.) This approach was used to identify a large number of members of a new family of transposable elements. These elements are similar to traditional DNA-mediated transposable elements in that they all have terminal inverted repeats (TIRs) (as opposed to retroelements that mediate transposition through RNA intermediates, Boeke J.D., D.J. Garfinkel, C.A. Styles and G.R. Fink 1985. Transposition of Ty elements mediated by RNA intermediates. Cell 40:491-500.). But unlike the transposable elements identified by classical genetic methods, these elements are relatively small and have no obvious protein-coding ability. These elements are known as miniature inverted repeat transposable elements or MITEs (Bureau et al. supra).

Since restriction fragment length polymorphism (RFLP) technique (Bostein D., R. White, M. Skolnick and R. W. Davis 1980. Construction of human genetic linkage map with restriction fragment length polymorphism. Am. J . Hum. Genet.32: 314-331.) Since being used as a tool for molecular mapping, genome mapping and fingerprinting technology has made great progress, manifested in other new technologies such as random amplified DNA polymorphism (RAPD, Welsh J. And M.McClelland 1990. Genome fingerprinting by PCR with random primers. Nucleic Acids Res. 18:7213-7218.; Williams J.G.K., A.R. Kubelik, K.J. Livak, J.A.Rafalski and S.V.Tingey 1990. Amplified DNA polymorphisms are effective genetic markers. Nucleic Acids Res.18:6531-6535.) and amplified fragment length polymorphisms (AFLP, Vos P., R. Hogers, M. Bleeker, M. Reijans, T. Van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper and M. Zabeau 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407- 4414.) Development. Recently, human DNA polymorphisms were detected using reverse transcription elements (Sinnet D., J.M.Deragon, L.R.Simard and D.Labuda1990. Alumorphs-polymerase chain reaction with Alu-specific primers. Genomics 7:331-334; and Nelson D.L., S.A. Ledbetter, L.Corbo, M.F.Victoria, R.Ramirez-Solis, T.D.Webster, D.H.Ledbetter and C.T.Caskey 1989. Alu polymerase chain reaction: a method for the rapid isolation of human-specific DNA sequences from composite DNA sources .Proc. Natl. Acad. Sci. 86:6686-6690.) and Simple Sequence Repeats (SSRs) (Litt M.And J. A. Luty 1989, by in vitro detection of dinucleotide repeats in the cardiac actin gene Hypervariable microsatellites revealed by amplification. Am.J. Hum. Genet. 44:397-401; Tautz D. 1989. Hypervariability of simple sequences as usual source of polymorphic DNA markers. Nucleic Acids Res. 17 : 6463-6471; and Weber J.L.And P.E.May 1989. A large number of types of human DNA polymorphisms that can be typed by polymerase chain reaction. Am.J.Hum.Genet.44:388-396.) Technology for the generation of new A generation of genome mapping and fingerprinting tools provides the conditions.

The restriction fragment length polymorphism (RFLP) labeling method includes digesting genomic DNA with restriction enzymes, separating DNA fragments by electrophoresis, and transferring the separated DNA fragments to a solid phase support composed of a nylon membrane, thereby obtaining A photocopy of a gel for hybridization to known DNA sequences. Known DNA sequences may be cloned genomic or cDNA sequences, or specific PCR products. The DNA sequence (probe sequence) is labeled with radioactive, fluorescent or colorable nucleotides. The hybridization results can be visualized by exposing the solid support to X-ray sensitive film, or when labeled with colored nucleotides, directly on the solid support. Depending on the source of the probe sequence, one or several DNA bands are often observed. Restriction fragment length polymorphisms can be shown as differences in banding patterns between different genotypes, which reflect differences in the distribution of a given restriction site.

The random amplified polymorphic DNA (RAPD) labeling method involves the use of short DNA sequences containing 10 nucleotides as primers to initiate PCR amplification using genomic DNA as a template. The nucleotide composition of the oligonucleotide primers is chosen randomly without reference to any existing DNA sequence. PCR products can be directly observed on agarose gel electrophoresis. Generally, 1-15 amplified DNA bands can be observed in the eukaryotic genome as amplification products. Polymorphisms can be directly detected by staining agarose gels, which are differences in the amplification patterns of different genotypes, and polymorphisms can reflect changes in single nucleotides or insertions/deletions in primers.

The amplified fragment length polymorphism (AFLP) labeling method involves digesting genomic DNA with restriction enzymes, combining the resulting genomic DNA fragments with an adapter sequence (a short double-stranded DNA sequence containing at one end the cause the same restriction site) ligation, use the oligonucleotide sequence homologous to the linker sequence as a primer for PCR amplification. The amplification result can be directly observed on the stained acrylamide gel, which is several (not more than 60) DNA fragments. The polymorphism of different genotypes can be expressed as the presence or absence of specific amplification bands, similar to RFLP, reflecting the difference in the distribution of a given restriction enzyme site and another group of genomic DNA.

The simple sequence repeat (SSR) marking method includes using a simple DNA sequence repeat (such as (TA)n, (CAGA)n, (GA)n, etc., "n" is generally 5-18) as a probe to identify an organism Gene clones carrying these simple sequence units in the gene library. Then, the isolated clones are sequenced, and a pair of primer sequences located at both ends of the SSR are designed for amplifying the SSR and the DNA sequences at both ends thereof. Polymorphisms of different genotypes appear as one or a few amplified DNA bands with one or a few nucleotide differences, reflecting differences in the number of repeats ("n") in simple sequences.

DNA markers based on retroelements and other long repetitive elements involve the design of primers located at both ends of the elements, the presence or absence of which in different genotypes can be considered polymorphisms.

Other types of DNA markers also exist, but they are combinations of the above DNA marker types. For example, CAP is enzyme-cleaved amplified polymorphic DNA, wherein PCR products are subjected to enzyme digestion after amplification. The primer pair can be designed according to the repeat element and AFLP primer or the sequence of different repeat elements.

There is an urgent need to provide a new universal nucleic acid sequence for linkage studies and fingerprint studies.

There is also an urgent need to provide a method for detecting polymorphisms in eukaryotes using the above-mentioned novel ubiquitous nucleic acid sequences.

Summary of Invention

One of the objects of the present invention is to provide a new universal nucleic acid sequence for linkage studies and fingerprinting studies.

Another object of the present invention is to provide a method for detecting polymorphisms in eukaryotes using the novel universal nucleic acid sequence.

According to the present invention, there is provided a method for detecting polymorphisms in target nucleic acid sequences, the method comprising the steps of:

(a) amplifying the target nucleic acid sequence with a pair of primers, wherein the first primer is homologous to a miniature inverted repeat transposable element (MITE), or a fragment thereof or a derivative thereof, and the first primer is homologous to the target nucleic acid sequence The MITE is annealed, and the second primer can be identical or different from the first primer, and can be homologous or non-homologous to the MITE sequence;

(b) fragments of the nucleic acid sequence of interest amplified in the isolation step (a);

(c) Analyzing the relationship between the fragment obtained in step (b) and the control fragment, the control fragment is obtained by amplifying the nucleic acid sequence with at least one primer pair capable of determining the difference between the fragment obtained in step (b) and the control fragment, the difference That is, it represents the polymorphism of the target nucleic acid.

In addition, according to the present invention, there is provided a method for genotyping eukaryotes, the method comprising the steps of:

(a) amplifying the target nucleic acid sequence in the eukaryote with a pair of primers, wherein the first primer is homologous to MITE, or a fragment thereof or a derivative thereof, and the primers may be compatible with the target nucleic acid sequence in the eukaryote The MITE anneals, and the second primer can be identical or different from the first primer, and can be homologous or not homologous to MITE;

(b) isolating the target nucleic acid sequence amplified in step (a);

(c) comparing the fragment obtained in step (b) with the fragment of the control nucleic acid sequence in the eukaryote, the fragment obtained in step (b) is consistent with the fragment of the control nucleic acid sequence, which means that the eukaryote has the nucleic acid sequence.

In addition, according to the present invention, there is provided a method for performing fingerprint analysis on eukaryotic organisms, the method comprising the steps of:

(a) using a pair of primers to amplify the nucleic acid sequence of eukaryotes, wherein the first primer is homologous to MITE, or a fragment thereof or a derivative thereof, and the primer is specific to the MITE sequence, and the second primer may be identical to the first primer or different, and may be of the same or different origin as MITE;

(b) isolating fragments of the nucleic acid sequence amplified in step (a), whereby the fragments isolated are representative of the eukaryote.

A preferred amplification step is achieved by PCR. The first primer was derived from the consensus sequence of the MITE elements. More preferably, the first primer contains a consensus sequence derived from Tourist, Stowaway, Barfly or Mariher.

Most preferably, the first primer contains a nucleic acid sequence selected from the group consisting of 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, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35.

Optionally, the second primer can be selected from the following primers: MITE-specific primers, primers based on SSR sequences, primers based on sequences of reverse transcription elements, primers based on the cloned nucleic acid sequences used for detection of RFLP, primers based on random genome sequences , primers based on vector sequences and primers based on gene sequences.

Further according to the present invention there is provided the use of polymorphisms when combined with the methods of the present invention for tracking eukaryotic progeny, determining hybridization of eukaryotic organisms, identifying linked phenotypic traits in eukaryotic organisms To identify the offspring individuals with desired genetic traits contributed by parental donors and/or parental recipients, or the offspring individuals with genetic markers that can be used as genetic maps for construction of genetic maps.

The method of the present invention can be used to isolate the genomic DNA sequence near the coding region or non-coding region of the gene. The genomic DNA sequence near the coding region of the gene is preferably a promoter or regulatory sequence.

In addition, according to the present invention, there is provided a nucleic acid fragment or its derivative, which is obtained by amplifying the nucleic acid sequence of eukaryotic organisms by using at least one primer homologous to MITE as a probe for the nucleic acid sequence.

Nucleic acid fragments or derivatives thereof are useful in marker-based screening (MAS), mapping-based cloning, heterozygote identification, fingerprinting, genotyping, and allele-specific markers.

Eukaryotes are preferably plants, animals or fungi.

The present invention also provides a method for genome mapping, comprising the steps of:

(a) fractionating the genome of a eukaryote;

(b) cloning the isolated genome into a vector;

(c) detecting the vector by amplifying the DNA in the cloned vector with a first primer homologous to a miniature inverted repeat transposable element (MITE) and a second primer, wherein the first primer is compatible with the DNA Miniature inverted repeat transposable element (MITE) hybridization, the second primer can be identical or different from the first primer, and homologous or nonlogous to the MITE sequence;

(d) separating the extension products in the amplification step according to size;

(e) determining the spectrum of the extension product;

(f) Genome reconstruction from overlapping maps.

The present invention also provides a method for mapping polymorphic genetic markers, comprising:

(a) providing a restriction enzyme digestion mixture of nucleic acid sequences in a biological sample from a eukaryote;

(b) amplifying the nucleic acid sequence mixture digested with restriction enzymes with a first primer and a second primer homologous to a miniature inverted repeat transposable element (MITE) or a fragment thereof or a derivative thereof, wherein the first primer is specific for For MITE, the second primer and the first primer can be identical or different, and the same MITE sequence can also be homologous or different;

(c) identifying a set of differentially amplified nucleic acid sequences in the mixture;

(d) performing specific genetic polymorphism mapping on at least one of said differentially amplified nucleic acid sequences, thereby providing a polymorphic marker.

The MITE-based labeling system of the present invention is different from any previously reported method, and has the characteristics of being simpler, capable of obtaining more information and having good reproducibility.

According to the purpose of the present invention, the following terms are defined.

The term "MITE" refers to Miniature Inverted Repeat Transposable Element. In fact, MITEs are a superfamily of transposable elements. These elements are less than 3 kb in length, contain complete or degenerate terminal inverted repeats flanked by target-site repeats of less than or equal to 10 base pairs, and are moderately or abundantly distributed across the genome.

MITEs are preferably less than 1 kb in length, contain complete or degenerate inverted terminal repeats flanked by TA or TAA target site repeats, and are moderately or abundantly distributed across the genome.

The term "MITE-based primer" refers to a primer comprising MITE or a fragment thereof, or a primer derived from MITE and capable of recognizing, hybridizing to or annealing to MITE.

The term "MITE-based genetic marker" (MGM) refers to a marker that hybridizes to a MITE element, or a genetic marker obtained by amplifying a nucleic acid sequence with at least one MITE primer and another primer, optionally another MITE Primers, primers based on SSR sequences, reverse transcription element sequences, RFLP sequences or gene sequences.

The term "inter-MITE polymorphism" (IMP) refers to a group of MGMs, referring to markers obtained by PCR amplification of a nucleic acid sequence using one or two different MITE primers.

The term "eukaryote" refers to plants, animals and fungi.

The term "homologous" refers to the ability of a nucleic acid sequence to hybridize to its complementary "homologous" nucleic acid sequence under stringent conditions in the context of homologous nucleic acid sequences.

Legend brief description

Figure 1 illustrates the results of the PCR products of TEM-4/-10 primer combination and individual TEM-10 on the agarose gel;

Fig. 2 illustrates that in a preferred embodiment of the present invention, the PCR product amplified by the TEM-1 primer labeled with IRD700 ^TM fluorescent dye is used on the 6% acrylamide gel to observe the result with LI-COR automatic system 4200, wherein P1 is the parent H. Vulgare, Lina (P1), P2 is the parent H. Spontaneum, Canada Park (P2), and the isolated individual comes from the cross of Lina and Canada Park DH (double haploid) population.

Figure 3 illustrates the results on the agarose gel of the PCR product amplified by the TEM-3/-10 primer combination under the long extension condition of 1 minute and 15 seconds;

Figures 4A and 4B illustrate the agarose gel results of different PCR products amplified by the TEM-1/-4 primer combination under extension conditions of 60 seconds and 75 seconds;

Figure 5 illustrates the linkage map of the hybridization of H.Vulgare cv.Lina and H.Spontaneum Canada Park populations, which shows the distribution of IMP sites detected with TEM-1 and TEM-10 primers;

Figure 6 illustrates the fingerprints of 27 Hordeum varieties on an agarose gel;

Figure 7 illustrates a part of the results of the fingerprint analysis of 27 Hordeum varieties with ^IRD700TM fluorescent dye-labeled TEM-1 primers;

Figure 8 illustrates the dendrogram obtained by clustering the genetic similarity matrix of 27 cultivars with UPGMA according to the band patterns of TEM-1 and TEM-10.

Figures 9A, 9B, 9C and 9D illustrate the broad application of MITE-based markers in different eukaryotes, showing the use of the main primer TEM-12 (Figure 9A), the main primer TEM-1 (Figure 9B), the main primer TEM -10 (Fig. 9C) and the master primer TEM-11 (Fig. 9D) were used for PCR amplification of DNA from 11 different sources;

Figures 10A, 10B, 10C, 10D and 10E illustrate an example of the results obtained with the master primer TEM-1 and its corresponding anchor primer.

Detailed description of the invention

The present invention provides a novel genetic marker, referred to herein as a MITE-based genetic marker (MGM). In this new approach, PCR is used to reveal polymorphisms by designing primers based on a large number of transposable elements, MITEs. The use of these transposable element-based primers was investigated by the segregation map of a barley double haploid mapping population and the genotyping of 26 barley (Hordeum Vulgare) cultivars and 1 Hordeum Spontaneum line Determination. According to the present invention, there is provided a novel class of DNA markers, referred to herein as MITE-based genetic markers, as well as the chromosomal location of these markers, their generality, diversity and fingerprinting results. Finally, we discuss the feasibility and conclusions of the MGM and IMP methods of the present invention.

Advantages and improvements over existing technologies

As mentioned above, MITE members are often linked to genes and thus are not restricted to repetitive sequence regions. This generality of MITE has enormous value. It shows that virtually any region in the genome of most eukaryotes can undergo IMP amplification.

A total of 50-100 scored bands could be amplified with each single primer, indicating that MITE exists in a high copy number in the genome. Based on 50-100 sites per primer, the whole genome can be easily screened with a few primers.

MITE primers can be used in combination with other types of primers, such as primers specific for SSRs, RT elements, sequenced RFLPs, random genomic sequences, vector sequences, and genes. This will increase the capabilities of the MGM method of the present invention.

The method of the present invention, in combination with the high-resolution LI-COR automated fluorescence genotyping system, has excellent efficacy in DNA mapping and fingerprinting. Its advantages over RAPD and RFLP in terms of power and resolution are obvious, since it can detect more sites in a single reaction. MGM and IMP analysis is simple, fast and low cost. Compared with RAPD analysis, the number of primers required is significantly reduced. Unlike AFLP and RFLP, MGM and IMP do not require restriction enzyme digestion or adapter ligation.

Technical Description

i) Plant material

The mapping population used consisted of 88 doubled haploids from a cross between the barley variety Lina and the H. Spontaneum variety Canada Park. This population has been used to construct linkage maps mainly based on RFLP markers.

A total of 27 cultivars (see Table 1) were used in the fingerprinting experiments, including 26 barley cultivars and one H. Spontaneum, Canada Park, which was used with Lina as a parent to generate the mapping population. The collection method includes two types of 2 rows and 6 rows. In the 2-row type include spring and winter varieties. A total of 27 breeds used had all been previously genotyped by RFLP, so RFLP-based genetic relationships among these breeds were known.

Table 1

Breeds used in fingerprint studies No. Species used1 Lina#05682 Canada Park3 Alexis4 Angora5 Ariel6 Azhul7 Ellice8 Express9 Fillipa10 Goldie11 Golf12 High amylose glacier13 Igri14 Ingrid15 Kinnan16 Maud17 Meltan18 Mentor19 Mette20 Mona21 Roland22 Saxo23 Svani54Treus2

ii) PCR detection system

Both detection systems were utilized to compare the precision and efficiency of polymorphism identification. The first system is the conventional agarose detection system. In this system, PCR is performed with conventional (unlabeled) primers. PCR products were visualized on a 2% agarose gel with or without Nusieve agarose (2/3 Nusieve: 1/3 regular agarose). The second system is the LI-COR automated DNA sequencing/genotyping system. Primers used in this system were labeled with IRD700 ^™ fluorescent dye (LI-COR, Inc., Licoln, Nebraska). PCR products were visualized on a 6% acrylamide denaturing gel using a 41 cm glass plate apparatus. Gel electrophoresis using LI-COR 4200 system.

iii) PCR

Seven master (Master) primers (Table 2) and their 3' anchor derivatives (Table 3) were designed and evaluated in this study. 6 kinds of main primers are MITE primers (TEM-1, TEM-2, TEM-3, TEM-10, TEM-11, TEM-12.), TEM-4 is several Tyl/copia-like reverse transcription to reverse transcription Conserved sequence segment of the reverse transcriptase (RT) domain of the transposon (Hirochika H. and r. Hirochika 1993. Tyl-copia-like retrotransposons are ubiquitous in plant genomes. Jpn. J. Genet. 68: 35-46.). The master primers are degenerate primers because there is the possibility of more than one nucleotide at certain positions. The anchor primer was a primer with nucleotides added outside the 3' end of the main primer (Table 3). MITE primers were designed based on the consensus sequence of the terminal inverted repeat (TIR) region of each MITE. Both TIRs were used to design primers. TEM-4 was only used in combination with other primers. These primers are used in both the agarose gel detection system and the LI-COR automatic detection system, except that the latter primers are labeled with fluorescent dyes.

Table 2

Source and sequence of primary primers

Primer Transposon Host Species MITE TIR Number Sequence

TEM-1 Stowaway Hordeum 44 (AG)TATTT(TA)GGAACGGAGGGAG

vulgare (SEQ ID NO: 1)

TEM-3 Tourist Triticum. 2 TT(TG)CCCAAAAGAACTGGCCC

aestivum (SEQ ID NO: 2)

TEM-10 Barfly H.vulgare 7 TCCCCA(CT)T(AG)TGACCA(CGT)CC

(SEQ ID NO: 3)

TEM-4 Tyl/copia§ Conserved RT NA GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TG

(SEQ ID NO: 4)

TEM-11 Barfly H.vulgare 8 TC(CT)CCATTG(CT)G(AG)CCAGCCTA

(SEQ ID NO: 5)

TEM-2 Tourist H.vulgare 4 CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCC

(SEQ ID NO: 6)

TEM-12 HsMarl(Ma Homo sapiens 58 AATT(CA)(CT)TTTTGCACCAACCT

riner)/MAD (SEQ ID NO: 7)

E1

§Hirochika and Hirochika 1993

table 3

The main theme and the corresponding anchoring primer TAM-1 (AG) TATTT (TA) GGAACGGGGGGGGGGAG SEQ ID NO: 1TEM-(AG) TATTT (TA) GGAACGGGGAGAGAGA SEQ ID NO: 8TEM-1C (AG) GGAACGGGGGAGC SEQ ID NO: 9TEM-1G (AG)TATTT(TA)GGAACGGAGGGAGG SEQ ID NO: 10TEM-1T (AG)TATTT(TA)GGAACGGAGGGAGT SEQ ID NO: 11TEM-2GGT CCTT(CT)TAA(AC)( )CCC SEQ ID NO: 6TEM-2A CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCA SEQ ID NO:12TEM-2C CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCC SEQ ID NO: 13TEM-2G CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCG SEQ ID NO:14TEM-2T CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCT SEQ ID NO:15TEM- 3      TT(TG)CCCAAAAGAACTGGCCC                SEQ ID NO：2TEM-3A     TT(TG)CCCAAAAGAACTGGCCCA               SEQ ID NO：16TEM-3C     TT(TG)CCCAAAAGAACTGGCCCC               SEQ ID NO：17TEM-3G     TT(TG)CCCAAAAGAACTGGCCCG               SEQ ID NO：18TEM-3T     TT (TG)CCCAAAAGAACTGGCCCT SEQ ID NO: 19TEM-4 GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TG SEQ ID NO: 4TEM-4A GT(TC)TT(ACGT)AC(GA)TCCAT(TC )TGA SEQ ID NO: 20TEM-4C GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGC SEQ ID NO: 21TEM-4G GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGG SEQ ID NO: 22TEM-4T GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGT SEQ ID NO: 23TEM-10 TCCCCA(CT)T(AG)TGACCA(CGT)CC SEQ ID NO: 3TEM- 10A TCCCCA(CT)T(AG)TGACCA(CGT)CCA SEQ ID NO: 24TEM-10C TCCCCA(CT)T(AG)TGACCA(CGT)CCC SEQ ID NO: 25TEM-10G TCCCCA(CT)T(AG)TGACCA (CGT)CCG SEQ ID NO: 26TEM-10T TCCCCA(CT)T(AG)TGACCA(CGT)CCT SEQ ID NO: 27TEM-11 TC(CT)CCATTG(CT)G(AG)CCAGCCTA SEQ ID NO: 5TEM- 11A TC(CT)CCATTG(CT)G(AG)CCAGCCTAA SEQ ID NO: 28TEM-11C TC(CT)CCATTG(CT)G(AG)CCAGCCTAC SEQ ID NO: 29TEM-11G TC(CT)CCATTG(CT)G (AG)CCAGCCTAG SEQ ID NO: 30TEM-11T TC(CT)CCATTG(CT)G(AG)CCAGCCTAT SEQ ID NO: 31TEM-12 AATT(CA)(CT)TTTTGCACCAACCT CA ) SEQ ID NO: 7TEM-12A( TT (CT) TTTTTGCACCCCTA SEQ ID NO: 32TEM-12C AATT (CA) (CT) TTTTTGCACCACCACCTC SEQ ID NO: 33TEM-12G AATT (CA) (CT) TTTTTGCACCACCTG SEQ ID NO: 34TEM-12TGC (CT) ttttttttttttttttttttttttttttttttttttttt for (CT) ID NO: 35

PCR amplification for the agarose detection system was done in a 25 μl volume containing 2.5 mM MgCl ₂ , 0.4 mM dNTPs, 1 μM of each primer and 0.625 units of AmpliTaq ^™ DNA polymerase (Perkin-Elmer). The following amplification program was used: the initial denaturation step was 94°C for 1 minute and 30 seconds, followed by a cycle of 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 1 minute, for a total of 35 cycles, and finally at 72°C. Extend for 5 minutes. This procedure was used unless otherwise specified. When TEM-1 is included, the annealing temperature is 60°C.

PCR amplification for the LI-COR detection system used the same conditions as the conventional agarose system, except that the total volume was 20 μl and 0.5 unit of AmpliTaq ^™ DNA polymerase (Perkin-Elmer) was used. The same amplification conditions were used (including TEM-1 with no temperature change). PCR amplification is performed in two steps. The first step is 35 cycles of preamplification with unlabeled primers. Take 3 μl of the pre-amplification mixture for the second step of amplification. Labeled primers were used in the second amplification step at a concentration of 0.1 μM (compared to 1 μM unlabeled primers in the first amplification step).

iv) Data collection and statistical analysis

a) Fingerprint and Genetic Similarity Analysis

For each individual, polymorphic bands and shared bands were scored as presence (1), absence (0) and missing data (9), respectively. The raw data obtained were determined by the determination method 2n of Nei and Li (nei M.And W. Li 1979. Studying the mathematical model of genetic variation with restriction endonuclease. Proc.Natl.Acad.Sci.76:5269-5273.) _xy /(n _x +n _y ), to generate relative genetic similarity (GS) matrices (matrices), where n _x and n _y are the number of bands of varieties x and y, respectively, and n _xy is the number of bands shared by both belt number. Both polymorphic bands and consensus bands were used to calculate GS values.

Dendrograms were generated from the GS matrix using the Unweighted Pairwise Arithmetic Mean Method (UPGMA). Analysis with two MITE primers (TEM-1 and TEM-10) resulted in a combined dendrogram. Then use the normalized Mantel statistical method (Mantel N.A, 1967, The detection of disease clustering and a generalized regression approach), CancerRes. 27: 209-220) to compare based on MITE The genetic similarity matrix obtained from the genetic markers and the genetic similarity matrix based on 313 polymorphic RFLP marker bands. Significance tests were performed by comparing the Z values observed for the distribution of 1000 randomly permuted matrices. All statistical analyzes were performed with NTSYS-pc software (Rohlf. F.J. 1994. NTSYS-pc Digital Structure and Multivariate Analysis System, Version 1.80, Exeter Software, N.Y.).

b) Genetic Mapping

By mapping the MITE-based genetic markers obtained with TEM-1 and TEM-10 primers within the framework of 71 RFLP markers that were used to construct hybrid maps of the barley cultivars Lina and H. Spontaneum Canada Park groups, to locate them. The 88 doubled haploid individuals within this population were used to map. Segregation ratios were analyzed by x2 analysis. Mapping was performed using the computer program MAPMAKER (Lander E.S., P.Green, J.Abrahamson, A.Barlow, M.J.Daly, S.E.Lincoln and L.Newburg 1987. MAPMAKER: A Basic Genetic Linkage for Constructing Experimental and Natural Populations An interactive software package for graphs. Genomics 1:174-181.). With an LOD threshold of 4, MITE-based genetic markers were assigned to linkage groups using a two-point analysis, with the exception of the 7H group, which formed two groups at that threshold, linked according to the published positions of the RFLP markers. At an LOD threshold of 2, the markers were assigned to linkage groups using multipoint analysis.

result

To evaluate MITE sequences in the PCR-based method of the present invention, primers were designed from the terminal inverted repeat (TIR) region, all primers oriented outwards from the TIR. In this way, any sequence amplified by these primers can be expected to lie between two adjacent MITEs within an amplifiable distance. These primers were used alone or in combination for segregation and analysis of 88 double haploid individuals of the hybrid progeny of barley variety Lina and H.Spontaneum Canada Park.

a) Single primer amplification

On an agarose gel, each MITE primer yielded approximately 10 score bands, of which 2-5 were polymorphic bands. These polymorphisms were clearly detected between the barley parents Lina and H. Spontaneum Canada Park, mostly in the expected Mendelian segregation ratio of 1:1 in the doubled haploids. Figure 1 shows an example of a separation profile.

M stands for λPstI marker. Lane 1 contains the PCR product of barley variety Lina. Lane 2 contains the PCR product of H. Spontaneum Canada Park. Lanes 3-28 contain PCR products for individuals in the mapping population.

Primer TEM-1 bands showed a high background, some of which were weak or barely observable, probably due to many closely related sequences, such as those resulting from variations in the TIR region. In this case, 2% formamide was added to the reaction mixture, because formamide has been reported to reduce PCR background and increase specificity (Nagaoka T.And Y.Ogihara 1997. The application of simple sequence repeat polymorphism in wheat and its role as Application of DNA markers in comparison of RFLP and RAPD markers. Theor. Appl. Genet. 94:597-602).

A total of about 100 scoring bands can be detected with primer TEM-1 on LI-COR sequencing gel, 60-70 with primer TEM-10, and 30-40 with TEM-3. A portion of polyacrylamide gel electrophoresis performed with TEM-1 is shown in FIG. 2 . Similar to the agarose gel assay, polymorphisms were clearly detected between the barley parent cultivar Lina and the H. Compare.

Lane 1 contains the PCR product of the parental barley variety Lina. Lane 2 contains the PCR product of the parental H. Spontaneum variety Canada Park. Lanes 3-45 contain the PCR products of individual individuals in the hybrid population of Lina and Canada Park.

b) Primer combination

Primer combination experiments are for gel detection systems only. Several situations were encountered when these primers were used in different combinations. The pattern of TEM-4/TEM-10 primer combination is consistent with that produced by using only TEM-10 primer, but the pattern of TEM-1/TEM-10 primer combination is different, and the pattern that appears when TEM-10 primer is used alone Most of the bands were suppressed, and the bands that appeared with TEM-1 alone were also less visible, and bands of smaller size appeared. The TEM-3/TEM-10 primer combination and longer extension time (1 min 15 sec) produced a different profile with 3 clearly visible separate bands that were different from each primer alone belt (Figure 3). Lanes 1-16 are individuals in the mapping group. No parents shown.

A similar situation was also observed in the case of using TEM-1 primer. When TEM-1 was combined with TEM-4, the pattern of bands (Fig. 4A) was unchanged, whereas the pattern was changed when this primer was combined with TEM-3 or TEM-10. In addition, the TEM-1/TEM-4 primer combination produced a larger separated band with a longer extension time (1 min 15 s), while some other bands were suppressed (Fig. 4B). Interestingly, this larger band (referred to as T1-4AA after the primer combination) was nearly identical to the segregation pattern of T4-10A (Fig. 1). T1-4AA and T4-10A are not identical products of the consensus primer TEM-4, since TEM-10 alone can also amplify the T4-10A band. In addition, T1-4AA is about 300bp longer than T4-10A. This indicates that the two primer combinations amplify tightly linked regions of DNA. This was to be expected since these transposable elements were predicted to be very high in copy.

The legends of FIGS. 4A and 4B are the same as those of FIG. 1 . The lane numbers of Figures 4A and 4B correspond to each other.

Some primer combinations produced inconsistent results. A possible interpretation is;

Each primer alone can amplify more than 10 bands, so combinations of these primers either produce too many bands to be clearly seen, or produce band patterns that vary with subtle changes in conditions;

Different annealing temperatures (such as when using TEM-4, which has a much lower annealing temperature) may be an important factor in determining the band pattern;

Different affinities of the primers may lead to the predominance of certain bands, as was the case with TEM-1 and TEM-10.

However, in a single-primer reaction, the use of a fluorescently labeled detection system may solve some of these problems, and the combination of primers may also significantly increase the number of detectable sites.

c) Chromosomal mapping of MITE-based genetic markers

Using the agarose detection system, 3 MITE primers and Tyl/copia retrotransposon primers yielded 15 detectable polymorphic markers in the mapping population. All markers had the expected 1:1 segregation ratio except for 2 of them. Thirteen of these markers were localizable on the map. The other 2 are not chained. This is a marker that deviates significantly from the expected separation ratio and may consist of two bands of similar size that cannot be resolved on the gel.

In Figure 5, MITE-based genetic markers are indicated in larger font and bold. Only the sites detected with fluorescently labeled TEM-1 and TEM-10 could be visualized on the acrylamide gel. Sites in parentheses indicate those that could not be placed at positions with LOD values greater than or equal to 2. Primers TEM-1 and TEM-10 can detect about 120 and 90 clear bands on LI-COR sequencing gel, respectively. The detected bands range in size from 100 bp to 1 kb. Figure 2 shows part of the results of the products amplified with TEM-1 on polyacrylamide gel.

Primers TEM-1 and TEM-10 could detect 75 and 19 polymorphic bands, respectively. Multiple pairs of amplified bands exhibit codominant behavior (T1-0.2 loci on 1H, T1-4 and T1-16 loci on 2H, T10-6 loci on 3H, T1-8 loci on 4H point and T1-36 site on 7H, Figure 5), but the rest of the bands showed a presence/absence pattern, exactly 41 bands came from the Lina parent, and 41 bands came from the H.spontaneum parent. Of the 70 mapped TEM-1 loci, 24 deviated significantly from the expected 1:1 segregation ratio. Except for one site (T1-19 site on 7H), the remaining 23 sites were located in the region where the RFLP markers of this mapping group also showed abnormal segregation ratio. Of the 18 TEM-10 sites, 2 sites deviated significantly from the expected segregation ratio, and they also localized in those regions where the RFLP markers also deviated from the expected 1:1 ratio.

A total of 88 loci were mapped. These loci covered all 7 linkage groups (Fig. 5). Furthermore, the distribution of these sites did not cluster significantly and was not, as expected, near centrioles where recombination typically occurs less frequently (eg 1H, 3H and 7H, Figure 5). In fact, this distribution is similar to the cDNA results detected by RFLP (L.S.O'Donoughue, unpublished). This suggests that MITEs are located in the genome containing the coding region and that it is possible to cover the entire genome with a limited set of MITE-based primers.

d) Fingerprint Analysis

A total of 27 cultivars (Table 1) including barley parent cultivar Lina and H. Spontaneum parent cultivar Canada Park and 25 barley cultivars were used to evaluate the use of these MITE primers in fingerprinting. On agarose, primers and primer combinations TEM-3, TEM-10, TEM1/TEM-3 and TEM-1/TEM-4 effectively discriminated between these species. 1-3 polymorphic bands were observed with each of these primers or combinations of primers. Figure 6 shows an example of a fingerprinting experiment on agarose.

The 3 separation bands in Fig. 6 indicated that 27 varieties were divided into 7 groups. M represents the molecular weight marker λPstI. The numbers correspond to those in Table 1.

Two MITE primers, TEM-1 and TEM-10, were studied in fingerprint analysis with a fluorescent label detection system. TEM-1 had a total of 62 scoring bands, 37 of which were polymorphic bands, while the remaining 22 were the same in all 27 varieties. Figure 7 shows a part of the results of this electrophoresis. TEM-10 had a total of 60 scoring bands, 34 of which were polymorphic bands, while the remaining 26 were common across all 27 cultivars. The species in Figure 7 can be found in Table 1.

Lanes 1-27 represent Lina, Canada Park, Alexis, Angora, Ariel, Azhul, Ellice, Express, Fillipa, Goldie, Golf, High amylose glacier, Igri, Ingrid, Kinnan, Maud, Meltan, Mentor, Mette, Mona, Roland , Saxo, Svani, Tellus, Tofta, Trebon and Vixen results. Dashes indicate markings that distinguish at least one variety from the others.

The GS matrix was obtained from the combined data of TEM-1, TEM-10 and the two primers using the coefficients of Nei and Li (Nei and Li, supra). Use the same data to generate a dendrogram. The dendrogram obtained from the combined data of TEM-1 and TEM-10 is shown in Figure 8. The dendrogram clearly separates H. Spontaneum cultivars from barley cultivars. With the exception of Azhul (6-row type), spring two-row types are grouped together, separated from the 4 winter types (Angora, Express, Igri and Vixen) included in this invention. The High Amylose Glacier variety that gathers with the two-row type in winter is the six-row type. The comparison between the GS matrix and the previous matrix obtained by RFLP analysis shows that there is a good positive correlation between the two, and the Mantel statistical value Z=0.69475. This positive correlation is very significant at probability P=0.0020, the Z value can be obtained randomly.

e) Universality of primers

To determine the generality of the primers, animal-derived and plant-derived MITE master primers were used on plant, insect and human genomic DNA.

Figure 9A, 9B, 9C and 9D show the typical results of the PCR amplification profiles of 11 different DNA sources, the main primer TEM-12 (Figure 9A), the main primer TEM-1 (Figure 9B), the main primer TEM- 10 ( FIG. 9C ) and the main primer TEM-11 ( FIG. 9D ), please refer to Table 2. The sources of DNA (listed for each lane above) are:

1) Normal human DNA, male.

2) Normal human DNA, female.

3) Human DNA, male albinism.

4) Human DNA, female albinism.

5) Insects: Trichogramma.

6) Legumes: soybeans.

7) Legumes: alfalfa.

8) Cruciferous plants: Canola.

9) Cereals: Wheat.

10) Cereals: oats.

11) Cereals: barley.

These results clearly demonstrate that MITE-based markers can be used in a wider range of species.

f) Diversity of sequences derived from the master sequence

To determine the diversity of the MITE-based tagging system, an extra nucleotide was added to the 3' end of the master primer sequence. This can increase the specificity of the amplified product, and is especially useful in cases where the amplification of the main sequence appears to be too complex to interpret, as was the case with the TEM-1 primer from Stowaway.

Figures 10A, 10B, 10C, 10D and 10E illustrate an example of the results obtained using the main primer TEM-1 in the preamplification step and its corresponding anchor primer listed in Table 3 in the amplification step. These figures show a comparison of the amplification patterns of cereal DNA (barley) obtained by polymerase chain reaction (PCR) with different primers, including TEM-1 alone (Fig. Add an extra "A" at the end (Fig. 10B), anchor primer TEM-1C, add a "C" (Fig. 10C), anchor primer TEM-1G, add a "G" (Fig. 10D), anchor primer TEM-1T, add a "T" (Fig. 10E).

These results clearly demonstrate that simpler amplification profiles can be obtained when A, C, T or G are anchored at the 3' end of TEM-1. It is also clearly shown that different but complementary amplification patterns can be obtained with different 3' end anchors.

General Purpose and Commercial Applications

Various studies have shown that transposable elements are present in virtually every species studied so far. Retrotransposons exist in high copy numbers in plant genomes. There are estimated to be 5×10 ⁵ copies of Alu family sequences in each haploid human genome, and an average of 5 kb of DNA can be translated into an Alu element. This element accounts for 5% of the primate genome (Berg DE and MM Howe 1989. Mobile DNA. Washington, American Society of Microbiology). The Tyl/copia group element has a maximum of 10 ⁶ copies per genome in Vicia species, accounting for more than 2% of the genome, but the variation between different species is relatively large (Pearce SR, H.Gill, D.Li, JSHeslop-Harrison, A. Kumar and AJ Flavell 1996, The Tyl-copia retrotransposon population in Vicia species: copy number, sequence heterogeneity, and chromosomal localization. Mol. Gen. Genet. 250:305-315). The BARE-1 retrotransposon contains 3×10 ⁴ copies, accounting for 6.7% of the barley genome (Suoniemi A., K. Anamthawat-Jonsson, T Arna and AHSchulman 1996. The retrotransposon BARE-1 is barley ( Hordeum vulgare L.) a major component dispersed in the genome. Plant Molecular Biology 30: 1321-1329). In the study of SanMiguel et al. (SanMiguel P., A. Tikhonov, y.-k. Jin, N, Motchoulskaia, D. Zakharov, A. Melake-Berhan, PS Springer, KJ Edwards, M. Lee, Z. Avramova and JL Benenetzen 1996 . Nested retrotransposons within intergenic regions in the maize genome. Science 274:765-768), sequencing of the 280 kb contiguous region flanking the maize Adhl-F gene isolated on a yeast artificial chromosome (YAC) revealed , there were 37 types of nested retrotransposon repeats, accounting for more than 60% of the clone.

Tourist and Stowaway elements (Bureau T.E.And S.R.Wessler 1992.Tourist: a large family of small molecule inverted repeat elements closely linked to maize genes. Plant Cell 4:1283-1294; and Bureau T.E.and S.R.Wessler 1994.Stowaway: a A new family of inverted repeat elements closely linked to monocot and dicot genes. Plant Cell 6:907-916) are members of the TIR-like transposable elements, although they are similar to traditional TIR transposable element families such as Ac and En/Spm makes a big difference. Barfly, a new member of the TIR transposable element family like Tourist and Stowaway, has been found to be linked to the barley xylan isomerase gene. These elements, together with some other elements of this type, are referred to as MITEs (Bureau T.E., P.C. Ronald, and S.R. Wessler 1996. A computer-based systematic survey revealed the presence of a large number of small inverted repeat elements in wild-type rice genes. Proc .Natl.Acad.Sci.93:8524-8529.), they have been found in a large number of plant species studied so far. MITEs are also expected to contain numerous copies in eukaryotic genomes.

The ubiquity and distribution of transposable elements in the genome allows their development as a PCR-based mapping tool. In fact, Sinnet et al. (Sinnet D., J.M.Deragon, L.R.Simard and D.Labuda 1990. Alumorphs-Using Alu-specific primers for polymerase chain reaction to detect human DNA polymorphisms. Genomics 7:331-334) utilized Alu-specific primers to find polymorphisms in different human DNA samples. These authors clearly showed that the use of these polymorphisms (termed Alu polymorphisms (alumorphs)) as a genomic analysis tool is feasible (Sinnet et al. supra), and successfully used these alumorphs to detect an alumorph associated with a Linkage mapping by simultaneous screening of multiple polymorphic loci using Alu oligonucleotide-directed PCR. Proc. Natl. Acad. Sci. 89:8448-8451). A copia-like retrotransposon PDR1 has also been successfully used to study polymorphisms and to identify different strains in Pisum by combining with other specific primers (Lee D., T.H.N.Ellis, L.Turner, R.P.Hellens and W.G. Cleary 1990. Copia-like elements in Pisum suggest that scattered repeats can be used for genetic analysis. Plant Molecular biology 15:707-722).

In the present invention, the TIR transposable element member MITE was used as a mapping and fingerprinting tool in barley, and successfully used conventional agarose system and LI-COR automatic DNA analysis system to detect polymorphisms and localize these MGMs in Fingerprinting of cultivars within existing genetic linkage maps and within barley species.

In a conventional agarose system, we show that 15 clearly scoreable polymorphisms can be detected using 3 MITE primers and a retrotransposon primer, 13 of which map to 4 linkage groups in barley . Each MITE primer or combination of primers can produce more than 10 scoring bands, of which 2-5 bands are polymorphisms. In the LI-COR automated DNA analysis system, each of the two MITE primers produced nearly 100 scored bands, of which up to 75 were polymorphic. All 7 barley linkage groups were marker mapped with these two primers. This suggests that MITE-based markers are randomly distributed across the genome.

New MITEs are continuously discovered using computer-based sequence similarity searches. As the number of MITEs increases, virtually any species with a high copy of MITEs can easily construct a detailed linkage map based solely on MGMs. Linkage studies of important genes can also be easily performed using MGM. The high level of variation detected between different cultivars of the same species with these MITE-based primers indicated that these primers have practical application value.

Some transposable elements such as Alu (Berg and Howe, supra; Makalowski W., G.A. Michell and D. Labuda 1994. Alu sequences in mRNA coding regions: sources of protein variation. Trends Genet. 10:188-193), and small Murine B2 elements (Clemens M.J. 1987. Potential role of RNA transcribed from B2 repeats in regulating mRNA stability. Cell 49:157-158) have been found to be closely linked to genes. MITE members are also frequently identified within plant and other eukaryotic genes. Stowaway was first discovered as a mutation factor at the maize wx site (Bureau and Wessler, supra). More than 100 genes have been found to contain MITEs in their coding or non-coding regions (Bureau et al., supra). The close linkage of retroelements to animal and plant genes, and the close linkage of MITEs to genes in crops and other plants provides new ways to identify genes or gene sequences. In fact, other types of transposable elements have been used in isolating gene sequences (Nelson D.L., S.A. Ledbetter, L. Corbo, M.F. Victoria, R. Ramirez-Solis, T.D. Webster, D.H. Ledbetter and C.T. Caskey 1989. Alu polymerase chain React: A method for the rapid isolation of human-specific DNA sequences from complex DNA sources. Proc.Natl.Acad.Sci.86:6686-6690; Souer E., F. Quattrocchio, N. De Vetten, J. Moland R. Koes 1995. A general method for isolating genes using high-copy transposable elements as markers. Plant Journal 7:677-685), genome analysis (Hirochika H. 1997. Rice retrotransposons: their regulation and role in Applications in Genome Analysis. Plant Molecular Biology 35: 231-240; Lee D., T.H.N. Ellis, L. Turner, R.P. Hellens and W.G. Cleary 1990. Copia-like elements in Pisum suggest that scattered repeats can be used for genetic analysis. Plant Molecular biology 15:707-722), Analysis of gene structure and expression (White S.E., L.F.Habera and S.R.Wessler 1994. Retrotransposons in gene flanking regions of normal plants: role of copia-like elements in the evolution of gene structure and expression .Proc.Natl.Acad.Sci.91:11792-11796).

The method of the invention can be used in several ways.

1) In linkage studies

a) Linkage maps can be constructed using MITE markers as described in this application. This requires an isolate as well as multiple parents. Linkage maps were constructed from segregation.

b) Linkage analysis can also be performed on phenotypic traits or genes. This can be done by performing a large number of separation analyses. In this case, polymorphic markers will be identified using two parents and two populations with phenotypic (traits) or genetic (genes) differences by PCR amplification with MITE primers, and thus presumed linkage.

c) According to the same principle, various statistical analyzes can be used, such as: one-point analysis of variance (ANOVA), interval plotting and composite interval plotting, to detect whether MGM or IMP is the same as quantitative trait loci (QTL). Association under complex genetic control.

d) If the association of important crop traits with markers is known, these markers can be used in marker-based screening (MAS), thereby accelerating breeding.

2) In fingerprint research

MGM and IMP methods can be used to help construct large insert fragment libraries, such as: YAC (Yeast Artificial Chromosome) and BAC (Bacterial Artificial Chromosome), which can facilitate species identification, gene isolation and marker switching.

a) The generated MGM and IMP markers can be used as markers for contig alignment and chromosome walking.

b) MGM and IMP methods can be easily used to fingerprint varieties and breed strains to determine their pedigree and genetic relationship, determine the contribution of parents to daughter lines, and identify new lines and varieties.

The MGM method can facilitate gene isolation and subcloning of genomic sequences.

a) When genes are tagged with transposable elements, MGMs can be exploited by taking advantage of their ubiquitous distribution in the genome. MITE primers can be used in conjunction with primers designed from tagged transposons. Flanking sequences can be amplified, which can then be used to isolate the wild-type gene. Compared with the conventional transposon-marked gene cloning method, this method saves one round of DNA library screening.

b) In a similar situation to gene isolation, MGM can be used to isolate sequences flanking known gene sequences. The flanking sequences can be amplified by PCRs using MITE primers and primers designed according to known gene sequences.

c) The RFLP marker can be converted to a PCR-based marker by amplification with primers from DNA clones detecting RFLP and MITE primers.

Although the above content is a description of the present invention in conjunction with specific embodiments, those skilled in the art should understand that the present invention can also be further modified, and this application should include any changes and applications that follow the principles of the present invention. and adjustments, which shall include improvements that meet the following conditions: technical solutions that fall into the known or conventional practice in the technical field of the present invention, technical solutions that have the above-mentioned basic features of the present invention, and fall within the following claims range of technical solutions.

Sequence Listing

<110> McGill University (MCGILL UNIVERSITY)

DNA LANDMARKS INC.

Benoit Landry (LANDRY, Benoit)

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Claims (20)

1. A method for detecting polymorphisms in target nucleic acid sequences, the method comprising the steps of: a) amplifying the nucleic acid sequence of interest with a pair of primers, wherein the first primer is homologous to a miniature inverted repeat transposable element (MITE), a fragment thereof or a derivative thereof, and the first primer may be homologous to the nucleic acid sequence of interest The MITE in MITE anneals, and the second primer can be the same as or different from the first primer, and can be homologous or not homologous to the MITE sequence; b) separating fragments of the nucleic acid sequence of interest amplified in step a); c) Analyzing the relationship between the fragment obtained in step b) and a control fragment obtained by amplifying the nucleic acid sequence with at least one primer for determining the difference in nucleic acid sequence between the fragment obtained in step b) and the control fragment , the difference represents the polymorphism of the target nucleic acid. 2. A eukaryotic genotyping method, the method comprising the steps of: a) using a pair of primers to amplify the target nucleic acid sequence in the eukaryote, wherein the first primer is homologous to MITE, or a fragment thereof or a derivative thereof, and the primer can anneal to the MITE in the target nucleic acid sequence, and the second The primer may be the same or different from the first primer, and be homologous or not to MITE; b) separating the fragments of the nucleic acid sequence amplified in step a); c) comparing the fragment obtained in step b) with the fragment of the control nucleic acid sequence in the eukaryote, wherein the identity of the fragment in step b) and the fragment of the control nucleic acid sequence is greater than that of the true nucleic acid sequence carrying the nucleic acid sequence present or absent in nuclear organisms. 3. A method for fingerprinting eukaryotes, the method comprising the steps of: a) amplifying a nucleic acid sequence in eukaryotes with a pair of primers, wherein the first primer is homologous to MITE, or a fragment thereof or a derivative thereof, the primer is specific to the MITE sequence, and the second primer can be the same as the first primer or Different, and homologous or different from MITE; b) isolating fragments of the nucleic acid sequence amplified in step a), such isolated fragments being representative of the eukaryote. 4. The method of claim 1, 2 or 3, wherein the amplifying step is performed by PCR. 5. The method of claim 1, 2, 3 or 4, wherein said first primer is derived from the consensus nucleic acid sequence of Tourist, Stowaway, Barfly or HSMarl Mariner and MADE1 elements. 6. The method in claim 1, 2, 3, 4 or 5, wherein said first primer contains a nucleotide sequence selected from the group consisting of: 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, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. 7. The method in claim 1, 2, 3, 4, 5 or 6, wherein said second primer is selected from the group consisting of: MITE-specific primers, primers based on SSR sequences, reverse transcription elements, detectable RFLP cloned nucleic acid sequence, random genome sequence, vector sequence or gene sequence. 8. Use of a polymorphism detectable by the method of claim 1, 2, 3, 4, 5, 6 or 7 for tracing the progeny of a eukaryotic organism. 9. Use of a polymorphism detectable by the method of claim 1, 2, 3, 4, 5, 6 or 7 for the determination of heterosis in eukaryotic organisms. 10. Use of a polymorphism detectable by the method of claims 1, 2, 3, 4, 5, 6 or 7 for identifying variation in linked phenotypic traits in eukaryotes. 11. Use of a polymorphism detectable by the method of claim 1, 2, 3, 4, 5, 6 or 7 as a genetic marker for constructing a genetic map. 12. Use of a polymorphism detectable by the method of claims 1, 2, 3, 4, 5, 6 or 7 in identifying hybrid progeny, wherein said progeny contain desired genetic characteristics of the body. 13. Use of the method of claim 1, 2, 3, 4, 5, 6 or 7 for isolating genomic DNA sequences adjacent to coding or non-coding sequences of a gene. 14. The method of claim 13, wherein the genomic DNA sequence adjacent to the coding sequence of the gene is a promoter or a regulatory sequence. 15. A nucleic acid fragment or derivative thereof obtained by amplifying a eukaryotic nucleic acid sequence with at least one primer homologous to MITE, wherein MITE is used as a probe on the nucleic acid sequence. 16. Use of the nucleic acid fragment or derivative thereof according to claim 15 for marker-based screening (MAS), mapping-based cloning, identification of heterozygotes, fingerprinting, genotyping and allele specificity mark. 17. The method of claim 2, wherein the eukaryote is a plant, animal or fungus. 18. The method of claim 3, wherein said eukaryote is a plant. 19. A genome mapping method, comprising the steps of: a) fractionating the genome of a eukaryotic organism; b) cloning the isolated genome into a vector; c) detecting the vector by amplifying DNA in the cloned vector with a first primer homologous to a miniature inverted repeat transposable element (MITE) and a second primer, wherein the first primer is compatible with the Miniature Inverted Repeat Transposable Element (MITE) hybridization in the DNA, the second primer can be the same or different from the first primer, and homologous or nonlogous to the MITE sequence; d) separating the extension products in the amplification step according to size; e) determining the spectrum of the extension product; f) Genome reconstruction from overlapping maps. 20. A method of mapping polymorphic genetic markers, comprising: a) providing a restriction enzyme digestion mixture of nucleic acid sequences in a eukaryotic sample; b) amplifying said restriction enzyme-digested nucleic acid sequence mixture with a first primer homologous to a miniature inverted repeat transposable element (MITE) or a fragment thereof or a derivative thereof, and a second primer, wherein the first primer is specific for For MITE, the second primer can be the same or different from the first primer, and be homologous or nonlogous to the MITE sequence; c) identifying a set of differentially amplified nucleic acid sequences in the mixture; d) mapping at least one of the differentially amplified nucleic acid sequences to a specific genetic polymorphism, thereby providing a marker for said polymorphism.

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