WO2000060113A2 - 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 another 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 TYPE OF TRANSPOSON-BASED GENETIC MARKER

BACKGROUND OF THE INVENTION

(a) Field of the Invention The invention relates to a method for genotyping a nucleic acid sequence using amplification with a primer pair comprising a first primer having a DNA sequence homologous to a miniature inverted- repeat transposable element (MITE) and a second primer, identical or different from the first primer. The invention generally relates to the use of MITE primers m fingerprinting or linkages studies.

(b) Description of Prior Art

After the discovery of the transposable element system Ac/Ds by McClintock (McClintock B. 1946. Maize genetics. Carnegie Inst . Wash. Yearbook 45: 176-186; and McClintock B. 1947. Cytogenetic studies of maize and neurospora . Carnegie Inst. Wash. Yearbook 46: 146- 152. ) , genetic identification of new transposable element systems (families) became a popular area of genetic studies m plants (Peterson P. A. 1986. Mobile elements m maize. Plant Breeding Reviews 4: 3-122.) as well as m other organisms. This was followed by the molecular characterization of transposable elements and exploitation of these elements as gene identification and isolation tools, especially after the cloning of the whi te locus with the copia retrotransposon m Drosophila (Bmgham P. M., R. Lewis and G. M. Rubin 1981. Cloning of DNA sequences from the white locus of D . elanogaster by a novel and general method. Cell 25: 693-704.), and molecular characterization of the maize transposable element Ac

(Pohlman R. F., N. V. Fedoroff and J. Messing 1984.

The nucleotide sequence of the maize controlling element 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 of Zea mays . EMBO J. 4: 17-25.) . Since then, transposable element-related studies have become a major focus in biological sciences.

As in other areas of biological research, the identification of transposable elements has been accelerated by modern computer technologies . Bureau et al . (Bureau T. E., P. C. Ronald, and S. R. Wessler 1996. A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild- type rice genes. Proc . Natl . Acad. Sci . 93: 8524- 8529.) adopted this approach to identify numerous members of a new family of transposable elements. These elements resemble the traditional DNA-mediated transposable elements (as opposed to retroelements which transpose via RNA intermediates, Boeke J. D., D. J. Garfinkel, C. A. Styles and G. R. Fink 1985. Ty elements transpose through an RNA internediate . Cell

40: 491-500.) in that they possess terminal inverted repeats (TIRs) . However unlike the classical genetically characterized transposable elements, these elements are small in size, and show no apparent coding capacity. These elements have been referred to as miniature inverted-repeat transposable elements or, MITEs (Bureau et al . supra) .

Since the introduction of the restriction fragment length polymorphism (RFLP) technique (Bostein D., R. White, M. Skolnick and R. W. Davis 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am. J. Hum. Genet. 32: 314-331.) as a molecular mapping tool, genome mapping and fingerprinting technologies have been advanced substantially as evidenced by the development of other new techniques such as randomly amplified DNA polymorphism (RAPD, Welsh J. and M. McClelland 1990. Fingerprinting genomes using PCR with arbitrary 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. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531-6535.), and amplified fragment length polymorphism (AFLP, Vos P., R. Hogers, M. Bleeker, M. Reiηans, T. van de Lee, M. Homes, A. Fπηters, J. Pot, J. Peleman, M. Kuiper and M. Zabeau 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acid Res. 23: 4407-4414.). The recently adopted techniques using retroelements (Sinnet D., J.-M. Deragon, L. R. Simard and D. Labuda 1990. Alumorphs-human DNA polymorphisms detected by polymerase chain reaction using A2u-specιfιc 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 rapid isolation of human- specifIC DNA sequences from complex DNA sources. Proc . Natl . Acad . Sci . 86: 6686-6690.) and simple sequence repeats (SSRs) (Litt M. and J. A. Luty 1989, A hypervariable microsatellite revealed by in vi tro amplification of a dmucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44: 397- 401; Tautz D. 1989. Hypervaπability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res. 17: 6463-6471; and Weber J. L. and P.E. May 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 388-396.) have set the stage for a new generation of genome mapping and fingerprinting tools. Restriction Fragment Length Polymorphism (RFLP) marker methodology consists of digesting genomic DNA with a restriction enzyme, separating the DNA fragments by electrophores, transferring the separated DNA fragments to a solid support consisting of a nylon membrane m order to obtain an image of the gel on a support that can be used for hybridization experiments with known DNA sequences . The known DNA sequence can be a cloned genomic or cDNA sequence or a specific PCR product. This DNA sequence (the probing sequence) is labeled with radioactive, fluorescent or colored nucleotides. Results of hybridization is seen by exposing the solid support to either an X-ray sensitive film or can be seen directly on the support when colored nucleotides are used to label the probe. One or a few DNA band is 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 difference m the distribution of a given restriction enzyme cutting sites.

Random Amplified Polymorphic DNA (RAPD) marker methodology consists of short DNA sequences of 10 nucleotides that are used as primers to drive a PCR reaction using total genomic DNA as template. The nucleotide composition of the oligonucleotide primers is chosen arbitrarily without any reference to existing DNA sequence. PCR products are visualized directly after agarose gel electrophoresis . Generally, one to 15 amplified DNA fragments can be seen as amplification product of an eukaryote genome. Polymorphisms are detected directly on an agarose gel after staining as differences m amplification patterns between genotypes and reflect single nucleotide changes m the primer and insertions/deletions. Amplified Fragment Length Polymorphism (AFLP) marker technology consists of digesting genomic DNA with a restriction enzyme, ligatmg the resulting genomic DNA fragments with an adapter sequence (a short double strand DNA sequence which has at one end the same sequence site as the one generated by the restriction enzyme used to digest the genomic DNA) and performing a PCR reaction using, as primer, an oligonucleotide homologous to the adapter sequence. Amplification results are visualized directly on an acrylamide gel after staining as several (up to 60) DNA fragments. Polymorphisms are seen as differences m the presence/absence of specific amplified DNA fragments m different genotypes and reflect, like RFLP, differences m the distribution of a given restriction enzyme cutting site but with a subset of the genomic DNA.

Simple Sequence Repeat (SSR) marker methodology consists of using a simple DNA sequence repeat (such as (TA)n, (CAGA)n, (GA)n, etc., "n" generally varying between 5 and 18) as probes to identify genomic clones from a gene library of an organism carrying these simple sequence motifs. The clones that are isolated are then sequenced and a pair of DNA primers surrounding the SSR are designed for PCR amplification of the SSR and the surrounding DNA sequences . Polymorphisms are seen as one or very few amplified DNA fragments varying by one or a few nucleotide differences m different genotypes and reflect differences m the number of repeats ("n") of the simple sequence.

DNA markers based on retroelements and other large repeated elements consist of designing primers surrounding the element and polymorphisms are found when the element is present or absent m different genotypes .

Other types of DNA markers exist but they are a combination of the types of DNA markers described above. For example, CAPs are cut amplified polymorphic

DNA where a PCR product is digested by restriction enzymes after PCR amplification. Primers pairs can be designed from a repeated element and an AFLP primer or from different repeated elements) . It would be highly desirable to be provided with a new pervasive nucleic acid sequence for use m linkage studies and m fingerprinting studies.

It would also be highly desirable to be provided with a method for detecting polymorphisms m eukaryotes using this new pervasive nucleic acid sequence .

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a new pervasive nucleic acid sequence for use m linkage studies and m fingerprinting studies.

Another aim of the present invention is to provide a method for detecting polymorphisms m eukaryotes using this new pervasive nucleic acid sequence.

In accordance with the present invention there is provided a method for detecting polymorphisms of a nucleic acid sequence of interest . The method comprises the steps of : a) amplifying said nucleic acid sequence of interest with a first primer homologous to a miniature inverted- repeat transposable element (MITE) , a fragment thereof or a derivative thereof, and a second primer wherein said first primer anneals with said MITE when present m said nucleic acid sequence of interest and said second primer is identical or not to the first primer, and homologous or not to a MITE sequence; b) separating fragments of the nucleic acid sequence of interest amplified m step a) ; and c) analyzing the fragments obtained m step b) m relation to reference fragments obtained from amplification of a nucleic acid sequence with the at least one primer for determining a difference m nucleic acid sequence between the fragments obtained m step b) and the reference fragments, whereby a difference is indicative of a polymorphism m the nucleic acid of interest . Also m accordance with the present invention, there is provided a method for genotypmg an eukaryote.

The method comprises the steps of : a) amplifying a nucleic acid sequence of said eukaryote with a first primer homologous to a MITE, a fragment thereof or a derivative thereof, and a second primer, wherein said first primer anneals with said MITE when present m said nucleic acid sequence of said eukaryote, and said second primer is identical or not to the first primer, and homologous or not to a MITE sequence ; b) separating fragments obtained from amplifying the nucleic acid sequence of step a) ; and c) comparing the fragments obtained from step b) with fragments of a reference nucleic acid sequence from said eukaryote, whereby identity of the fragments of step b) with the fragments of the reference nucleic acid sequence is indicative of said eukaryote having said nucleic acid sequence .

Further m accordance with the present invention, there is provided a method for 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 a MITE, a fragment thereof or a derivative thereof, and a second primer, wherein said first primer is specific for a MITE sequence and said second primer is identical or not to the first primer, and homologous or not to the

MITE sequence; and b) separating fragments obtained from amplifying the nucleic acid sequence of step a) , whereby the fragments so-separated are representative of the eukaryotic organism. Preferably the step of amplifying is effected by PCR procedures. The first primer is derived from a consensus sequence from a MITE element . More preferably, the first primer has a nucleic acid sequence derived from a consensus sequence from Tourist, Stowaway, Barfly, or Mariner.

Most preferably, the first primer has a nucleic acid sequences 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.

The second primer optionally is a primer selected from the group consisting of a MITE specific primer, a primer based on a SSR sequence, a primer based on a retroelement sequence, a primer based on a sequence of a cloned nucleic acid detecting a RFLP, a primer based on a random genomic sequence, a primer based on a vector sequence and a primer based on a gene sequence .

Also m accordance with the present invention, there is provided the use of a polymorphism as with the method of the present invention for tracing progeny of a eukaryotic organism, for determining hybridity of a eukaryotic organism, for identifying a variation of a linked phenotypic trait m a eukaryotic organism, for identifying individual progenies from a cross wherein said progenies have a desired genetic contribution from a parental donor and/or recipient parent, or as genetic markers for constructing genetic maps.

The method of the present invention may be used for isolating genomic DNA sequence surrounding a gene- codmg or non-coding DNA sequence. The genomic DNA sequence surrounding the gene-codmg DNA sequence is preferably a promoter or a regulatory sequence .

Further m accordance with the present invention, there is provided a nucleic acid fragment or a derivative thereof, obtained by amplifying a nucleic acid sequence of a eukaryotic organism with at least one primer homologous to a MITE for use as a probe on nucleic acid sequences.

The nucleic acid fragment or the derivative thereof may be used for marker-assisted selection (MAS) , map-based cloning, hybrid certification, fingerprinting, genotypmg, and allele specific marker.

The eukaryote or eukaryotic organism is preferably a plant, an animal or fungi.

Still m accordance with the present invention, there is provided a method for genome mapping, which comprises the steps of: a) fractionating the genome of a eukaryotic organism; b) cloning the genome so-fractionated into a vector; c) testing the vectors so-cloned by amplifying DNA m the vectors so-cloned using a first primer homologous to a miniature inverted- repeat transposable element (MITE) , and a second primer, the first primer being capable of hybridizing to a miniature inverted-repeat transposable element (MITE) m the DNA, and the second primer is identical or not to the first primer, and homologous or not to a MITE sequence; d) separating extension products of the amplification step by size; e) measuring the pattern of extension products; and f) reconstructing the genome from the overlapping patterns. Also m accordance with the present invention, there is provided a method for mapping a polymorphic genetic marker, which comprises: a) providing a mixture of restriction enzyme- digested nucleic acid sequences from a biological sample from a eukaryotic organism; b) amplifying the mixture of restriction enzyme-digested nucleic acid sequences using a first primer homologous to a miniature inverted-repeat transposable element (MITE) , a fragment thereof or a derivative thereof, and a second primer, wherein the first primer is specific for a MITE, and the second primer is identical or not to the first primer, and homologous or not to a MITE sequence; c) identifying a set of differentially amplified nucleic acid sequences m the mixture ; and d) mapping at least one of the differentially amplified nucleic acid sequences to a unique genetic polymorphism, thereby providing a marker for the polymorphism. The MITE-based marker system of the present invention is different from any of the approaches of the prior art, is much simpler, is more high informative and repeatable.

For the purpose of the present invention the following terms are defined below.

The term "MITE" is intended to mean a miniature inverted-repeat transposable element. In fact, MITEs are a superfamily of transposable elements. These elements are less than 3 kilobases long, contain perfect or degenerate terminal mverted-repeats, are flanked by a target site duplication of less than, or equal to 10 base pairs, and are moderately to highly abundant m the genome. MITEs are preferably less than one kilobases long, have perfect or degenerate terminal inverted repeats, are flanked by a TA or TAA target site duplication and are moderately to highly abundant in the genome .

The term "MITE-based primer" is intended to include a primer comprising a MITE or a fragment thereof, and a primer derived from a MITE and that recognizes a MITE, hybridizing or annealing thereto. The term "MITE-based genetic marker" (MGM) is intended to mean a marker hybridizing to a MITE element, or a marker produced by the PCR amplification of a nucleic acid sequence using at least one MITE primer and optionally another MITE primer or a primer based on a SSR sequence, a retroelement sequence, a RFLP sequence or a gene sequence .

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

The term "eukaryote" or "eukaryotic organism" is intended to refer to plants, animals and fungi.

The term homologous is intended to mean in the context of a homologous nucleic acid sequence, a nucleic acid sequence which would hybridize under stringent conditions to a complement of the nucleic acid sequence it is homologous with.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates PCR products of primer combination TEM-4/-10 or TEM-10 alone on an agarose gel;

Fig. 2 illustrates a section of the PCR results of IRD700™ fluorescence dye-labeled TEM-1 primer, visualized on a 6% acrylamide gel with the LI -COR automated system 4200 m accordance with a preferred embodiment of the invention, which PI is parent H. vulgare, Lma (PI) , P2 is parent H. spontaneum, Canada Park (P2), and the segregating individuals are from a cross between the Lma and Canada Park DH (Doubled Haploid) population;

Fig. 3 illustrates PCR results of TEM-3/-10 with longer extension time of 1 minute and 15 seconds on agarose gel;

Figs. 4A and 4B illustrate PCR results on agarose gel of TEM-1/-4 showing different products with a 60-second extension time and a 75-second extension time; Fig. 5 illustrates a linkage map of the H. vulgare cv. Lma x H. spontaneum Canada Park population showing the distribution of IMP loci detected with the TEM-1 and TEM-10 primers;

Fig. 6 illustrates a fingerprinting of the 27 Hordeum lines on agarose gel;

Fig. 7 illustrates a section of the fingerprinting result of 27 Hordeum lines with IRD700™ fluorescence dye-labeled TEM-1 primer;

Fig. 8 illustrates a dendrogram resulting from the UPGMA clustering of the genetic similarity matrix of 27 cultivars, based on the TEM-1 and TEM-10 banding patterns .

Figs. 9A, 9B, 9C and 9D illustrate the universal use of the MITE-based markers m different eukaryotes, showing PCR-amplifled profiles of eleven different sources of DNA using 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) Figs. 10A, 10B, IOC, 10D and 10E illustrate an example of the results obtained with the Master primer (TEM-1) and its corresponding anchored primer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new genetic marker referred to herein as MITE-based genetic marker

(MGM) . In this new method using PCR, polymorphisms are revealed with primers designed from the abundant transposable elements, MITEs . The usefulness of these transposable element -based primers was determined by studying segregation patterns m a barley doubled- haploid mapping population and m genotypmg 26 cultivars of Hordeum vulgare and one line of Hordeum spontaneum . In accordance with the present invention, there is provided a novel type of DNA markers, referred herein as MITE-based genetic markers, as well as the chromosomal localization of these markers, their universality and versatility and the fingerprinting results. Finally, we discuss the feasibility and the generalization of the MGM and IMP approaches of the present invention. Advantages and Improvements over Existing Technology

As mentioned above, MITE members are frequently found to be associated with genes, and thus, are not confined to repetitive regions. This pervasiveness of MITEs is of enormous value. It indicates that virtually any region of the genome is prone to IMP amplifications m most eukaryotic organisms. A total of 50-100 scorable bands were amplified with every single primer, indicating that MITEs are present m the genome m high copy numbers . With several primers and 50-100 loci per primer, the whole genome can be covered readily m the screening. The MITE primer can be combined with other types of primers such as primers specific for SSRs, retroelements, sequenced RFLPs, random genomic sequences, vector sequences, and genes. This will certainly increase the capacity of the MGM method of the present invention.

The method of the present invention, combined with high resolution LI -COR automated fluorescence genotypmg system, provides enormous power m DNA mapping and fingerprinting techniques. Its power and resolution over RAPD and RFLP are obvious as many more loci could be detected a single reaction. MGM and IMP analysis are easy, fast and cost effective. In contrast to RAPD analysis, significantly fewer primers are needed. Unlike the AFLP and RFLP techniques, MGM and IMP does not require digestions with restriction enzymes or adapter ligation. Technical Description I) Plant materials The mapping population used consists of 88 doubled-haploid individuals from a cross between Hordeum vulgare cultivar Lma and H. spontaneum cultivar Canada Park. This population has been used to construct a linkage map based mostly on RFLP markers. A total of 27 cultivars (see Table 1) were used m the fingerprinting experiments including 26 H. vulgare entries and one H. spontaneum entry, Canada Park, which was used together with Lma as parents to generate the mapping population. The collection included two-row and six-row types. Among the two-row types, both spring and winter cultivars were included. All 27 cultivars were previously used m an RFLP genotypmg study and therefore, the RFLP-based genetic relationships among these cultivars were known. Table 1

Cultivars used in the fingerprinting study

Identificati Cultivar used on Number

1 Lina #0568

2 Canada Park

3 Alexis

4 Angora

5 Ariel

6 Azhul

7 Ellice

8 Express

9 Fillip a

10 Goldie

11 Golf

12 High amylose glacier

13 Igri

14 Ingrid

15 Kinnan

16 Maud

17 Meltan

18 Mentor

19 Mette

20 Mona

21 Roland

22 Saxo

23 Svani

24 Tellus

25 Tofta

26 Trebon

27 Vixen

ii) PCR detection systems

Two detection systems were used to compare the resolution and efficiency in polymorphism identifications. The first was the regular agarose detection system. In this system, PCRs were performed with regular primers (non-labeled) . PCR products were visualized in 2% agarose gels, with or without Nusieve agarose (2/3 Nusieve : 1/3 regular agarose) . The second was the LI -COR automated DNA sequencing/genotyping system. Primers for this system were labeled with IRD700™ fluorescent dye (LI-COR, Inc., Licoln, Nebraska) . PCR products were visualized with 6% acrylamide denaturing gel with a device of 41 cm long glass plates. The gel electrophoresis was run with the LI-COR 4200 system, m) PCRs Seven master primers (Table 2) and their 3¹- anchored derivatives (Table 3) were designed and evaluated m this study. Six of the master primers were MITE primers (TEM-1, TEM 2, TEM-3, TEM-10, TEM-11 and TEM- 12) and TEM-4 was a segment of the conserved sequences of the reverse transcriptase (RT) domain of several Tyl/copia-like retrotransposons (Hirochika H. and R. Hirochika 1993. Tyl - copia group retrotransposons as ubiquitous components of plant genomes. Jpn. J. Genet. 68: 35-46.) . The master primers were degenerate as more than one nucleotide was possible m certain position. The anchored primers were the master primers with the additional nucleotide added at the 3' end of the master primer (Table 3) . MITE primers were designed from the consensus sequences m the terminal inverted repeats (TIR) regions of MITEs from each category. Both TIRs were used to design the primers. TEM-4 was used only m combinations with other primers . The primers were used on both the agarose gel detection system and LI-COR automated detection system except that primers for the latter were labeled with a fluorescent dye.

- l i

Table 2

Master Primers Sources and Sequences

Primer Transposon Host species No. of Sequence MITEs TIRs

TEM-1 Stowaway Hordeum 44 (AG)TATTT(TA)GGAACGGAGGGAG vulgare (SEQ ID N0.1 )

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 Ty1/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 HsMar1 (Ma Homo sapiens 58 AATT(CA)(CT) I I I I GCACCAACCT riner)/MAD

E1 (SEQ ID NO: 7)

Hirochika and Hirochika 1993

Table 3 Master primers and the corresponding anchored primers

TEM-1 (AG)TATTT(TA)GGAACGGAGGGAG SEQ ID NO:1

TEM-1A (AG)TATTT(TA)GGAACGGAGGGAGA SEQ ID NO:8

TEM-1 C (AG)TATTT(TA)GGAACGGAGGGAGC SEQ ID NO:9

TEM-1 G (AG)TATTT(TA)GGAACGGAGGGAGG SEQ ID NO:10

TEM-1T (AG)TATTT(TA)GGAACGGAGGGAGT SEQ ID NO:11

TEM-2 CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCC SEQ ID NO:6

TEM-2A CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCA SEQ ID NO:12

TEM-2C CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCC SEQ ID NO:13

TEM-2G CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCG SEQ ID NO:14

TEM-2T CCTT(CT)TAA(AC)(ACGT)GAACAA(CG)CCCT SEQ ID NO:15

TEM-3 TT(TG)CCCAAAAGAACTGGCCC SEQ ID NO:2

TEM-3A TT(TG)CCCAAAAGAACTGGCCCA SEQ ID NO:16

TEM-3C TT(TG)CCCAAAAGAACTGGCCCC SEQ ID NO:17

TEM-3G TT(TG)CCCAAAAGAACTGGCCCG SEQ ID NO:18

TEM-3T TT(TG)CCCAAAAGAACTGGCCCT SEQ ID NO:19

TEM-4 GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TG SEQ ID NO:4

TEM-4A GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGA SEQ ID NO:20

TEM-4C GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGC SEQ ID NO:21

TEM-4G GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGG SEQ ID NO:22

TEM-4T GT(TC)TT(ACGT)AC(GA)TCCAT(TC)TGT SEQ ID NO:23

TEM-10 TCCCCA(CT)T(AG)TGACCA(CGT)CC SEQ ID NO:3

TEM-10A TCCCCA(CT)T(AG)TGACCA(CGT)CCA SEQ ID NO:24

TEM-10C TCCCCA(CT)T(AG)TGACCA(CGT)CCC SEQ ID NO:25

TEM-10G TCCCCA(CT)T(AG)TGACCA(CGT)CCG SEQ ID NO:26

TEM-10T TCCCCA(CT)T(AG)TGACCA(CGT)CCT SEQ ID NO:27

TEM-11 TC(CT)CCATTG(CT)G(AG)CCAGCCTA SEQ ID NO:5

TEM-11 A TC(CT)CCATTG(CT)G(AG)CCAGCCTAA SEQ ID NO:28

TEM-11 C TC(CT)CCATTG(CT)G(AG)CCAGCCTAC SEQ ID NO:29

TEM-11 G TC(CT)CCATTG(CT)G(AG)CCAGCCTAG SEQ ID NO:30

TEM-11T TC(CT)CCATTG(CT)G(AG)CCAGCCTAT SEQ ID NO:31

TEM-12 AATT(CA)(CT)TTTTGCACCAACCT SEQ ID NO:7

TEM-12A AATT(CA)(CT)TTTTGCACCAACCTA SEQ ID NO:32

TEM-12C AATT(CA)(CT)TTTTGCACCAACCTC SEQ ID NO:33

TEM-12G AATT(CA)(CT)TTTTGCACCAACCTG SEQ ID NO:34

TEM-12T AATT(CA)(CT)TTTTGCACCAACCTT SEQ ID NO:35

PCR amplif ications for the agarose detection system were performed in a 25 μl volume containing 2 . 5 mM MgCl₂, 0.4 mM dNTP, 1 μM of each primer and 0.625 unit of AmpliTaq™ DNA polymerase (Perkin-Elmer) . The following profile was used: an initial denaturation step of 1 min 30 sec at 94°C; followed by 35 cycles of 30 sec at 94°C, 45 sec at 58°C and 1 min at 72°C; and a final extension of 5 min at 72°C. This profile was used unless otherwise indicated. An annealing temperature of 60°C was used whenever TEM-1 was included. PCR amplifications for the LI-COR detection system were performed with the same conditions as in the regular agarose system, except a total reaction volume of 20 μl and 0.5 unit of AmpliTaq DNA polymerase

(Perkin-Elmer) were used. The same general profile was used (without temperature change for TEM-1) . PCR amplifications were done in two steps. The first step is a preamplification with non-labeled primers for 35 cycles. An aliquot of 3 μl of the preamplification mix was used for the second step of amplification. A 0.1 μM concentration of the labeled primer was used in the second round of amplification (compared with 1 μM of non-labeled primer in the first step) . iv) Data collection and statistical analyses a) Fingerprinting and genetic similarity analyses

Polymorphic as well as common bands were scored as presence (1), absence (0), or missing data (9) for each individual . The resulting raw data matrices were used to generate relative genetic similarity (GS) matrices using Nei and Li's (Nei M. and W. Li 1979. Mathematical models for studying genetic variation in terms of restriction endonucleases . Proc . Natl . Acad. Sci. 76: 5269-5273.) measurement, 2n_xy/ (n_x + n_y) , where n and n_y are the numbers of bands in lines x and y, respectively and n_xy is the number of bands shared by both lines. Both polymorphic and common bands are used to calculate the GS values.

Dendrograms were generated based on the GS matrices using the unweighted pair-group method arithmetic average (UPGMA) . A combined dendrogram resulting from analyses with two MITE primers (TEM-1 and TEM-10) was generated. The normalized Mantel statistic (Mantel N. A. 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27: 209-220) was used to compare the genetic similarity matrix based on the MITE-based genetic markers with a genetic similarity matrix of the same cultivars based on 313 polymorphic RFLP marker bands. The test of significance was performed by comparing the observed Z-value with the distribution of 1000 random permutations of the matrices. All statistical analyses were performed with the NTSYS-pc software (Rohlf F. J. 1994. NTSYS-pc numerical taxonomy and multivariate analysis system, version 1.80, Exeter Software, N. Y.). b) Genetic mapping

The localization of the MITE-based genetic markers generated with the TEM-1 and TEM-10 primers was performed by mapping these within a framework of 71 RFLP markers that had been used previously to construct a map of the Hordeum vulgare cultivar Lina x H. spontaneum Canada Park population. A subset of 88 doubled haploid individuals of this population was used for the mapping. Segregation ratios were analyzed using χ² 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: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181.). The MITE-based genetic markers were assigned to linkage groups using two-point analysis at a LOD threshold of 4 with the exception of group 7H which formed two groups at this threshold and were linked based on published location of RFLP markers) . Multipoint analysis with a LOD threshold of 2 was used to place the markers within the linkage groups. RESULTS

To evaluate the MITE sequences in the PCR-based method of the present invention, primers were designed from the terminal inverted repeat (TIR) regions, with all primers being directed outward from the TIRs . In this way, any sequences amplified by these primers are expected to lie between two adjacent MITEs within amplifiable distances. These primers were used alone or in combinations in a segregation analysis using a doubled-haploid population of 88 individuals from a cross between Hordeum vulgare cultivar Lina and H. spontaneum cultivar Canada Park. a) Single primer amplifications

On agarose gels, each of the MITE primers, generated around 10 scorable bands with 2-5 being polymorphic. These polymorphisms were clearly detected between the H. vulgare parent Lina and the H. spontaneum parent Canada Park and mostly showed the expected 1:1 Mendelian segregation in the doubled- haploid. Fig. 1 shows an example of the segregation patterns .

M identifies λPstl marker. Lane 1 contains PCR products of H. vulgare Lina. Lane 2 contains PCR products of H. spontaneum Canada Park. Lanes 3-28 contain PCR products of individuals in the mapping population.

Primer TEM-1 showed a high background with some very weak to almost invisible bands, probably due to many closely related sequences, e.g., those resulted from variations in the TIR regions. In this case, 2% formamide was added to the reaction mixes, since formamide has been reported to reduce PCR background and enhance specificity (Nagaoka T. and Y. Ogihara 1997. Applicability of inter-simple sequence repeat polymorphisms in wheat and their use as DNA markers in comparison to RFLP and RAPD markers. Theor. Appl . Genet. 94 : 597-602) . A total of approximately 100 scorable bands were detected on the LI-COR sequencing gel with primer TEM-1, , between 60 and 70 with primer TEM-10 and between 30 and 40 could be detected with primer TEM-3. A section of the acrylamide gel electrophoreses with TEM-1 is shown in Fig. 2. As in the agarose detection system, the polymorphisms were clearly detected between the H. vulgare parent Lina and the H. spontaneum parent Canada Park and mostly showed 1:1 Mendelian segregation in the doubled-haploid population. Lane 1 contains PCR products from parent H . vulgare, Lina. Lane 2 contains PCR products from parent H. spontaneum, Canada Park. Lanes 3-45 contains PCR products from individuals of the population resulting from the cross Lina X Canada Park. b) Primer combinations

Primer combination tests were only carried out with the agarose detection system. Several situations were encountered when these primers were used in different combinations. Whereas the combination TEM- 4/TEM-10 yielded the same pattern as TEM-10 alone, the combination TEM-l/TEM-10 produced a different result, in which, the majority of bands from TEM-10 alone were inhibited, bands from TEM-1 alone were also less visible, and bands of smaller sizes appeared. Combination TEM-3/TEM-10 with longer extension time (1 mm 15 sec) , yielded a different pattern with three clearly visible segregating bands different from that of either primer alone (Fig. 3) . Lanes 1-16 are individuals m the mapping population. No parents are shown .

Similar situations were seen with primer TEM-1. Whereas the banding pattern did not change when TEM-1 was combined with TEM-4 (Fig. 4A) , the pattern did change when this primer was combined with TEM-3 or TEM- 10. Moreover, with a longer extension time (1 mm 15 sec) , the combination TEM-l/TEM-4 yielded a larger segregating band with some other bands suppressed (Fig. 4B) . Interestingly, the larger band (referred to as T1-4AA after the primer combination) segregated almost the same as band T4-10A (Fig. 1) . T1-4AA and T4-10A were not the same product of the common primer TEM-4 since TEM-10 alone also amplified band T4-10A. Also, T1-4AA was approximately 300bp larger than T4-10A. This indicates that the two primer combinations amplified tightly linked regions of DNA. This is not unexpected because these transposable elements are predicted to be present m high copy numbers.

Legends m Figs . 4A and 4B are the same as m Fig 1. Lane numbers correspond to each other m Figs . 4A and 4B.

Some primer combinations yielded inconsistent results. Possible explanations are:

Each primer alone amplified more than 10 bands and therefore, combinations of these primers could either yield too many bands to be clearly visualized or could yield band patterns that fluctuate with micro condition changes; Different annealing temperatures (as with TEM-4, which has a much lower annealing temperature) may be an important factor determining the pattern produced; and Different affinities of primers may result m the predominance of certain bands, as was the case with TEM-1 and TEM-10.

Nevertheless, it is likely that, as with the single primer reactions, using the fluorescence labeling detection system, some of these problems will be resolved and that primer combinations will significantly increase the number of detectable loci. c) Chromosome localization of MITE-based genetic markers

Using the agarose detection system, the three

MITE primers and the Tyl/copia retrotransposon primer generated a total of 15 detectable polymorphic markers on the mapping population. All except two, segregated m the expected 1:1 segregation ratio. Thirteen of these markers could be placed on the map. The other two markers remained unlinked. These were the markers exhibiting significant deviation from the expected segregation ratio and are likely to consist of two bands of similar size that could not be separated on agarose .

In Fig. 5, the MITE-based genetic markers are seen m a larger font and m bold character. Only the loci detected on acrylamide gel with the fluorescently labeled TEM-1 and TEM-10 can be seen. Loci parentheses are those that could not be placed with a LOD score greater than or equal to 2. Approximately 120 and 90 clear bands were detected on a LI-COR sequencing gel with primers TEM-1 and TEM-10, respectively. The size range of the bands detected was approximately 100 bp to 1 kb . Part of the amplification result with TEM-1 as visualized by polyacrylamide gel electrophoreses is shown in Figure 2.

Seventy- five and 19 polymorphic bands were generated with the TEM-1 and TEM-10 primers, respectively. Some pairs of bands exhibited co-dominant behavior (loci Tl- 0.2 on 1H, Tl-4 and Tl-16 on 2H, T10-6 on 3H, Tl-8 on group 4H and Tl-36 on 7H, Figure 5) , but the remaining bands exhibited a presence/absence pattern with exactly 41 coming from the Lina parent and 41 from the H. spontaneum parent. Of the 70 mapped TEM-1 loci, 24 significantly deviated from the expected 1:1 segregation ratio. All 24 loci except one (Tl-19 on 7H) mapped to areas where RFLP markers also exhibited distorted segregation ratios in this mapping population. Two of the 18 TEM-10 loci significantly deviated from the expected segregation ratio and these were again located in areas where RFLP loci also deviated from the expected 1:1 ratio. In total, 88 loci were mapped. These loci covered all seven linkage groups (Figure 5) . Furthermore, the distribution of the loci showed no significant clustering other than that which would be expected around centromeric regions where recombination is typically reduced (e.g., groups 1H, 3H and 7H, Figure 5) . In fact, the distribution is similar to that found with cDNAs detecting RFLPs (L. S. O'Donoughue, unpublished) . This suggests that MITEs are located in areas of the genome containing coding sequences and that it will be possible to cover the entire genome with a limited set of MITE-based primers, d) Fingerprinting

A total of 27 cultivars, which included the H. vulgare parent Lina, H. spontaneum parent Canada Park and 25 H. vulgare cultivars (Table 1) were used to assess the usefulness of these MITE primers in fingerprinting. On agarose, the primers and primer combinations TEM-3, TEM-10, TEM-1/-3 and TEM-1/-4 were found to be useful in distinguishing these cultivars. One to three polymorphic bands were seen with each of these primers and combinations. An example of the fingerprinting experiments on agarose is shown in Fig. 6.

The three segregating bands in Fig. 6 indicated that the 27 cultivars separated into 7 groups. M represents the molecular weight marker λPstl . The numbers correspond to those in Table 1.

Two MITE primers, TEM-1 and TEM-10 were studied in the fingerprinting analysis with the fluorescence labeling detection system. A total of 62 bands were scored for TEM-1, 37 of which were polymorphic, and the remaining 22 were the same across all 27 cultivars. A section of this electrophoresis is shown in Fig. 7. A total of 60 bands were scored with TEM-10, 34 of that were polymorphic and the remaining 26 were the same across all 27 cultivars. Identification of the lines of Fig. 7 can be found in Table 1.

Lanes 1-27 present the results from 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, respectively. Dashes indicate markers that distinguished at least one cultivar from others.

GS matrices were generated with TEM-1, TEM-10 as well as the combined data of both primers, using Nei and Li's coefficient (Nei and Li, supra) . Dendrograms were generated with the same sets of data. The dendrogram of the combined data of TEM-1 and TEM-10 is shown in Fig. 8. The dendrogram clearly separates^' the H. spontaneum line from the H. vulgare cultivars. With the exception of Azhul (six-row type) , the spring two- row types clustered together and separated from the 4 winter types (Angora, Express, Igri and Vixen) included m the present invention. The High Amylose Glacier line clustering with the winter two-rows is a six-row type. A comparison of the GS matrix with the one obtained earlier with an RFLP analysis showed a good correlation between the two, with a Mantel statistic of Z = 0.69475. This positive correlation was highly significant with a probability of P = 0.0020, that this value of Z would be obtained by chance alone. e) Universality of the primers

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

Figs. 9A, 9B, 9C and 9D show a typical result of PCR-amplifled profiles of eleven different sources of DNA using 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) , as referred to, Table 2. The sources of DNA (listed above each lane) are :

I) Normal human DNA, male. 2) Normal human DNA, female.

3) Human DNA, male with albinism.

4) Human DNA, female with albinism.

5) Insect: Trichogramma .

6 ) Legume : soya . 7) Legume: alfalfa.

8) Crucifer: Canola.

9) Cereal: wheat.

10) Cereal : oat .

II) Cereal: barley. These results clearly demonstrate that MITE-based markers can be used m a broad range of species. f) Versatility of the Master derived sequences To demonstrate the versatility of the MITE- based marker system, the Master primer sequences were modified by adding an additional nucleotide at their 3 ' end. This has the effect of increasing the specificity of the amplified product and is especially useful when the amplification profile generated by the Master sequence is too complex to interpret as with the TEM-1 primer derived from Stowaway.

Figs. 10A, 10B, IOC, 10D and 10E show an example of the results obtained with the Master primer TEM-1 m a preamplification step and its corresponding anchored primer listed m Table 3 m the amplification. The Figures shows polymerase chain reaction (PCR) - amplified profiles of cereal DNA (barley) comparing the profile obtained with Master primer TEM-1 alone (Fig. 10A) ; anchored primer TEM-1A, anchored with an additional "A" at its 3' end (Fig. 10B) ; anchored primer TEM-1C, is anchored with "C" (Fig. 10C) ; anchored primer TEM-1G, anchored with "G" (Fig. 10D) and; anchored primer TEM-1T, anchored with "T" (Fig. 10E) . It is clear from these results that a more simple amplification pattern is obtained when TEM-1 is anchored at its 3' end with either an A, C, T, or G. It is also clear that different and complementary amplification patterns are obtained with the different 3' end anchors.

General Purposes and Commercial Applications

Various studies have shown transposable elements to be present m virtually every species studied to date. Retrotransposons are present m plant genomes m high copy numbers. The Alu family was estimated to be 5 X 10⁵ copies per haploid human genome that translates to one Alu element m every 5 kb of DNA. This element alone accounts for 5% of the genome primates (Berg D. E. and M. M. Howe 1989. Mobile DNA. Washington, American Society of Microbiology) . Tyl/copia group elements can accumulate up to 10⁶ copies per genome m Vicia species, making up to >2% of the genome, although wide variations were seen across species (Pearce S. R., H. Gill, D. Li , J. S. Heslop- Harrison, A. Kumar and A. J. Flavell 1996. The Tyl - copia group retrotransposons Vi cia species: copy number, sequence heterogeneity and chromosome localisation. Mol . Gen. Genet. 250: 305-315). The BARE-1 retrotransposon has a copy number of 3 x 10⁴ and makes up to 6.7% of the barley genome (Suoniemi A., K. Anamthawat-Jonsson, T Arna and A. H. Schulman 1996. Retrotransposon BARE- 1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome. Plant Molecular Biology 30: 1321-1329). In the study by SanMiguel et al . (SanMiguel P., A. Tikhonov, Y.-K. Jin, N. Motchoulskaia, D. Zakharov, A. Melake-Berhan, P. S. Springer, K. J. Edwards, M. Lee, Z. Avramova and J. L. Bennetzen 1996. Nested retrotransposons m the mtergenic regions of the maize genome. Science 274: 765-768), sequencing of a contiguous 280-kb region flanking the maize Adhl-F gene isolated on a yeast artificial chromosome (YAC) clone revealed 37 classes of nested retrotransposon repeats that accounted for >60% of the clone.

The Tourist and Stowaway elements (Bureau T. E. and S. R. Wessler 1992. Touri st : A large family of small inverted repeat elements frequently associated with maize genes. Plant Cell 4: 1283-1294; and Bureau T. E. and S. R. Wessler 1994. Stowaway: A new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. Plant Cell 6: 907-916) are members of the TIR class of transposable elements, although they differ significantly from the traditional TIR transposable element families like Ac and En/Spm. Barfly, a new member of the TIR transposable elements like Tourist and Stowaway, is found to be associated with the barley xylose isomerase gene. These elements, together with some other elements of the type, collectively referred to as MITEs (Bureau T. E., P. C. Ronald, and S. R. Wessler 1996. A computer-based systematic survey reveals the predominance of small inverted-repeat elements m wild-type rice genes. Proc . Natl . Acad. Sci . 93: 8524-8529), were found m a great number of plant species studied so far. MITEs are also expected to be present m high copy numbers m eukaryotic genomes .

The ubiquity and dispersion throughout the genome of transposable elements suggest that they can be exploited as PCR-based mapping tools. Indeed, Smnet et al . (Smnet D., J.-M. Deragon, L. R. Simard and D. Labuda 1990. Alumorphs- -human DNA polymorphisms detected by polymerase chain reaction using Alu- specific primers. Genomics 7: 331-334) used Alu- specific primers m search of polymorphisms among different human DNA samples. These investigators clearly demonstrated the feasibility of using these polymorphisms (termed alumorphs) as a genome analysis tool (Smnet et al . , supra) and successfully used these alumorphs to detect the linkage of one alumorph to a human disease (Zietkiewicz E., M. Labuda, D. Smnet, F. H. Glorieux and D. Labuda 1992. 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 , was also successfully used to study polymorphisms and, m combination with other specific primers, to diagnose different lines m Pisum (Lee D., T. H. N. Ellis, L. Turner, R. P. Hellens and W. G. Cleary 1990. A copia-like element m Pisum demonstrates the uses of dispersed repeated sequences m genetic analysis. Plant Molecular Biology 15: 707- 722) . In the present invention, the TIR transposable element members, MITEs, are used as mapping and fingerprinting tools m barley and succeeded m both the regular agarose system and the LI-COR automated DNA Analysis system m detecting polymorphisms, localizing these MGMs into an existing genetic linkage map and fingerprinting cultivars within the H. vulgare species.

In the regular agarose detection system, we showed that with three MITE primers and one retrotransposon primer, 15 clearly scorable polymorphisms were detected and 13 of the 15 were mapped to four linkage groups of barley. Each MITE primer or primer combination generated more than 10 scorable bands with 2-5 being polymorphic. In the LI- COR automated genotypmg system, each of the two MITE primers shown generated close to 100 scorable bands with up to 75beιng polymorphic. Markers mapping to all seven barley linkage groups were obtained using these two primers. This demonstrates the random distribution of the MITE-based markers m genomes. New MITEs are constantly being uncovered by computer-based sequence similarity searches. As the number of MITEs increases, detailed linkage maps of virtually any species with high copy numbers of MITEs can be readily constructed based solely on MGMs. Linkage studies of important genes with MGMs can also be readily carried out. The high level of variation detected with these MITE-based primers among cultivars within the same species demonstrates the practical value of these primers. Some transposable elements, such as Alu (Berg and Howe, supra; Makalowski W., G. A. Michell and D. Labuda 1994. Alu sequeces in the coding regions of mRNA: a source of protein variability. Trends Genet. 10: 188-193), and the mouse B2 element (Clemens M. J. 1987. A potential role for RNA transcribed from B2 repeats in the regulation of mRNA stability. Cell 49: 157-158) , have been found to be frequently associated with genes. MITE members were also frequently identified within plant and other eukaryotic genes. Stowaway was first discovered as a mutation cause at the wx locus of maize (Bureau and Wessler, supra) . More than 100 genes were found to harbor MITEs in their coding or non-coding regions (Bureau et al . , supra) . The close association of retroelements with animal and plant genes, and MITEs with genes in agronomic crops and other plants has opened a new way of characterizing genes or gene sequences. Indeed, studies have been done 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 reaction: A method for 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. Mol and R. Koes 1995. A general 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 analysis. Plant Molecular Biology 35: 231-240; Lee D., T. H. N. Ellis, L. Turner, R. P. Hellens and W. G. Cleary 1990. A copia-like element m Pisum demonstrates the uses of dispersed repeated sequences in genetic analysis. Plant Molecular Biology 15: 707- 722) , and in analysis of gene structure and expression (White S. E., L. F. Habera and S. R. Wessler 1994. Retrotransposons in the flanking regions of normal plant genes: A role for copia-like elements in the evolution of gene structure and expression. Proc. Natl. Acad. Sci . 91: 11792-11796) using other types of transposable elements.

The applications of the method of the present invention are several folds. 1) In Linkage Studies a) As described in the present application, linkage maps can be constructed with MITE markers.

This requires a segregating population and the parents. Linkage maps are constructed based on the segregation. b) Linkage to a phenotypic trait or a gene can also be carried out. This can be accomplished in conjunction with bulked segregant analysis to expedite the investigation. In this case, two parents and the pools that are phenotypically (with a trait) or genetically (with a gene) distinct are to be used in PCR amplification with MITE primers to identify polymorphic markers and therefore putative linkages. c) By the same principle, the association of MGM or IMP with Quantitative Trait Loci (QTL) controlling traits under complex genetic control can be detected using various statistical analysis such as single point ANOVAs , Interval Mapping and Composite Interval Mapping. d) Once linkages of markers with traits of agronomical importance are known, these markers can be used in marker assisted selection (MAS) to expedite breeding programs. 2) In Fingerprinting Studies

The MGM and IMP approaches can be used to assist construction of large insert libraries such as YACs (yeast artificial chromosomes) and BACs (bacterial artificial chromosomes) , to assist m cultivar identifications and to assist gene isolation as well as for marker conversion. a) The MGM and IMP markers generated can serve as landmarks aligning contigs and m chromosome walking. b) The MGM and IMP approaches can be readily explored m fingerprinting cultivars and breeding lines to determine their pedigrees and genetic relationships, to determine the degree of contribution of a parent to progeny lines, and m certification of new lines and cultivars .

The MGM approach can be used to assist m gene isolations and subclonmg genomic sequences. a) When a gene is tagged with a transposable element, MGM can be exploited, by virtual of its pervasiveness the genome. A MITE primer can be used m conjunction with a primer designed from the tagging transposon. Flanking sequence can be amplified which can then be used to isolate the wild type gene. This approach can save one round of DNA library screening compared to regular cloning of a transposon tagged gene . b) With a similar scenario to gene isolation, MGM can be exploited to isolate genome sequences flanking known gene sequences. A MITE primer and a primer designed from the known gene sequence can be used m PCRs to amplify the flanking sequences. c) Amplification using a primer from a DNA clone detecting an RFLP used combination with a MITE primer may be used to convert an RFLP marker to a PCR based marker.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .

Claims

WHAT IS CLAIMED IS:

1. A method for detecting polymorphisms of a nucleic acid sequence of interest, said method comprising the steps of: a) amplifying said nucleic acid sequence of interest with a first primer homologous to a miniature inverted-repeat transposable element (MITE) , a fragment thereof or a derivative thereof, and a second primer wherein said first primer anneals with said MITE when present m said nucleic acid sequence of interest and said second primer is identical or not to the first primer, and homologous or not to a MITE sequence; b) separating fragments of the nucleic acid sequence of interest amplified m step a) ; and c) analyzing the fragments obtained m step b) m relation to reference fragments obtained from amplification of a nucleic acid sequence with said at least one primer for determining a difference m nucleic acid sequence between said fragments obtained m step b) and said reference fragments, whereby a difference is indicative of a polymorphism said nucleic acid of interest .

2. A method for genotypmg a eukaryote, said method comprising the steps of : a) amplifying a nucleic acid sequence of said eukaryote with a first primer homologous to a MITE, a fragment thereof or a derivative thereof, and a second primer, wherein said first primer anneals with said MITE when present m said nucleic acid sequence of said eukaryote, and said second primer is identical or not to the first primer, and homologous or not to a MITE sequence; b separating fragments obtained from amplifying the nucleic acid sequence of step a) ; and c) comparing said fragments obtained from step b) with fragments of a reference nucleic acid sequence from said eukaryote, whereby identity of the fragments of step b) with the fragments of the reference nucleic acid sequence is present or absent m said eukaryote having said nucleic acid sequence .

3. A method for fingerprinting a eukaryotic organism, said method comprising the steps of: a) amplifying a nucleic acid sequence of a eukaryotic organism with a first primer homologous to a MITE, a fragment thereof or a derivative thereof, and a second primer, wherein said first primer is specific for a MITE sequence and said second primer is identical or not to the first primer, and homologous or not to the MITE sequence; and b) separating fragments obtained from amplifying the nucleic acid sequence of step a) , whereby said fragments so- separated are representative of said eukaryotic organism.

4. The method of claim 1, 2 or 3 , wherein the step of amplifying is effected by PCR procedures.

5. The method of claim 1, 2, 3 or 4, wherein said first primer is derived from a consensus sequence from Tourist, Stowaway, Barfly or HSMarl Mariner and MADE1 element sequence.

6. The method of claim 1, 2, 3, 4 or 5, wherein said first primer has 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, and SEQ ID NO: 35.

7. The method of claim 1, 2, 3, 4, 5 or 6, wherein said second primer is selected from the group consisting of a MITE specific primer and a primer based on a SSR sequence, a retroelement sequence, a sequence of a cloned nucleic acid detecting a RFLP, a random genomic sequence, a vector sequence or a gene sequence .

8. Use of a polymorphism detected by the method of claim 1, 2, 3, 4, 5, 6 or 7 for tracing progeny of a eukaryotic organism.

9. Use of a polymorphism detected by the method of claim 1, 2, 3, 4, 5, 6 or 7 for determining hybridity of a eukaryotic organism.

10. Use of a polymorphism detected by the method of claim 1, 2, 3, 4, 5, 6 or 7 for identifying a variation of a linked phenotypic trait in a eukaryotic organism.

11. Use of a polymorphism detected by the method of claim 1, 2, 3, 4, 5, 6 or 7 as genetic markers for constructing genetic maps.

12. Use of a polymorphism detected by the method of claim 1, 2, 3, 4, 5, 6 or 7 for identifying individual progenies from a cross wherein said progenies have a desired genetic contribution from a parental donor and/or recipient parent.

13. The method according to claim 1, 2, 3, 4, 5, 6 or 7 for isolating genomic DNA sequence surrounding a gene-coding or non-coding DNA sequence.

14. The method of claim 13, wherein the genomic DNA sequence surrounding the gene-coding DNA sequence is a promoter or a regulatory sequence.

15. A nucleic acid fragment or a derivative thereof, obtained by amplifying a nucleic acid sequence of a eukaryotic organism with at least one primer homologous to a MITE for use as a probe on nucleic acid sequences.

16. Use of the nucleic acid fragment or a derivative thereof as defined in claim 15 for marker assisted selection (MAS) , map-based cloning, hybrid certification, fingerprinting, genotyping, and allele specific marker.

17. The method of claim 2, wherein the eukaryote is a plant, animal or fungi.

18. The method of claim 3, wherein the eukaryotic organism is a plant.

19. A method for genome mapping, comprising the steps of : a) fractionating the genome of a eukaryotic organism; b) cloning the genome so- fractionated into a vector; c) testing the vectors so-cloned by amplifying DNA in the vectors so-cloned using a first primer homologous to a miniature inverted- repeat transposable element (MITE) , and a second primer, said first primer capable of hybridizing to a miniature inverted-repeat transposable element (MITE) in the DNA, and said second primer is identical or not to the first primer, and homologous or not to a MITE sequence; d) separating extension products of the amplification step by size; e) measuring the pattern of extension products; and f) reconstructing the genome from the overlapping patterns .

20. A method for mapping a polymorphic genetic marker, said method comprising: a) providing a mixture of restriction enzyme- digested nucleic acid sequences from a biological sample from a eukaryotic organism; b) amplifying the mixture of restriction enzyme-digested nucleic acid sequences using a first primer homologous to a miniature inverted-repeat transposable element (MITE) , a fragment thereof or a derivative thereof, and a second primer, wherein said first primer is specific for a MITE, and said second primer is identical or not to the first primer, and homologous or not to a MITE sequence; c) identifying a set of differentially amplified nucleic acid sequences m the mixture; and d) mapping at least one of the differentially amplified nucleic acid sequences to a unique genetic polymorphism, thereby providing a marker for the polymorphism.