TWI414607B - Method to establish a molecular marker for detecting polymorphism in plant genome and the molecular marker establised thereby - Google Patents

Abstract

The invention relates to a method to establish a molecular marker for detecting polymorphism in plant genome. The method of the present invention comprises amplifying the genome by polymerase chain reaction with the utilization of the primers that specific to the first transposable element and the second transposable element, respectively, so that a fragment of the genome comprising the sequence between any first transposable element and any second transposable element, a fragment of the genome comprising the sequence between any two of the first transposable elements, and/or a fragment of the genome comprising the sequence between any two of the second transposable elements can be obtained, wherein the first transposable element and the second transposable element are different; and analyzing the polymorphism of the fragments to establish the molecular marker. The molecular marker established by the present invention is called inter-transposable elements polymorphism (ITEP) molecular marker. The invention also provides a molecular marker for detecting polymorphism in a plant genome.

Description

Method for establishing molecular markers for detecting plant genomic polymorphism by using plant jumpers and molecular markers established

The present invention relates to the technical field of molecular biology. In particular, the present invention relates to a molecular marker for detecting the polymorphism of a plant genome.

Transposable elements (TE) are widely present in the genome of eukaryotes. For example, jumpers account for about 45% of the human genome (Lander et al., 2001, Nature 409: 860-921); 30% (Goff et al., 2002, Science 296: 92-100; Jiang et al., 2004, Curr. Opin. Plant Biol. 7: 115-119); Arabidopsis 10% (Arabidopsis Genome Initiative 2000). In monocots with larger genomes (such as maize, barley), jumpers are up to 75% (SanMiguel et al., 1996, Science 274: 765-768; Kumar and Bennetzen, 1999, Annu. Rev. Genet. 33:479-532; Vicient et al., 1999, Plant Cell 11:1769-1784).

Each type of jumper includes a transcoded fragment and a non-coding fragment, wherein the transcoded fragment contains complete or partial open reading frames (ORFs), and the transcoded protein product Related to the transposition reaction. Most of the jumpers are inserted into the genome and produce a target-site duplication (TSD) at both ends of the insertion (Feschotte et al., 2002, Mobile DNA II. pp. 1147-1158, American Society for Microbiology, Washington, DC).

Jumpers are divided into two categories according to the different intermediates in their transposition:

The first class (class 1) is a retrotransposon, and the form of the intermediate is RNA. Such inverted position jumpers comprise (1) inverted-bit hoppings with long-length repeats (LTR) of long fragments, which are ubiquitous in the plant genome, based on the trans-transcription reverse transcriptase (reverse) Arrangement of transcriptase, RT) and integrase, divided into Ty1/like copia (Ty1/copia-like) inversion jumper and Ty3/gypsy (Ty3/gypsy-like) inversion jumper; 2) Non-LTR retrotransposons that do not have long fragments of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) (Kumar and Bennetzen, 1999, Annu. Rev. Genet. 33:479-532).

The second class (class 2) is a transposon, and the form of the intermediate is DNA. Based on the similarity of such inverted bit jumper transcoding product sequences, it is divided into (1) helitrons; (2) short terminal inverted repeat transposon (TIR transposon), and its transcoding product The two ends of the jumper are combined, and then the two ends are cut and then transposed (Capy et al., 1998, Nat. Rev. Genet. 3:329-341; Craig et al., 2002, Mobile DNA II. American Society for Microbiology, Washington, DC; Zhang et al, 2004, Genetics 166: 971-986), the terminal inverted repeat jumper comprises Tc1/mariner, hAT, CACTA, Mutator-like elements (MULEs) and PIF/Pong; And (3) miniature-inverted repeat transposable elements (MITEs), which do not contain transcoded fragments (Feschotte et al., 2002, Mobite DNA II. pp. 1147-1158, American Society for Microbiology, Washington, DC).

DNA molecular marker technology has been widely used in molecular biology. The earliest established technique is called restriction fragment length polymorphism (RFLP) (Bostein et al., 1980, Am. J. Hum. Genet. 32:314-331), but the DNA samples required for this technique are numerous, time consuming and labor intensive, and isotopes are also required. However, since the development of the polymerase chain reaction, there are many DNA molecular markers derived, such as: (1) Random amplified polymorphic DNA (RAPD), which can be used to analyze the entire genome. Randomly synthesized 10mer primers, using different varieties of DNA as templates, polymerase chain reaction, random synthesis of template DNA by primers, each variety can produce polymorphic DNA products, which can be used as molecular markers (Williams) Et al., 1990, Nucleic Acids Res. 18:6531-6535); (2) simple sequence repeat (SSR), because some 1 to 8 repetitive sequences can be found in the genome, (GA) _n , (TAC) _n, etc., these repetitive sequences are adjacent to each other, and the number of sets is highly variable during the evolution process. Therefore, primers can be designed in the sequence retention region at both ends of the repetitive sequence group, which can be solved by polymerase chain reaction technology. Detection of polymorphism (Tautz, 1989, Nucleic Acids Res. 17:6463-6471); (3) Inter-simple sequence repeat (ISSR), which explores the interval between two adjacent SSRs More area (4) Amplified fragment length polymorphisms (AFLP), a fingerprint technique developed by Vos et al. in 1995 (Vos et al., 1995, Nucleic Acids Res. 23: 4407-4414) It combines polymerase chain reaction with restriction enzyme hydrolysis technology. However, this method requires detection by fluorescence or radioisotope.

Because of its wide distribution, jumpers can be applied as molecular markers. The earliest reported are Alu repetitive sequences spread throughout the human genome. They design specific primers for Alu sequences and perform polymerase chain reaction to obtain molecular markers (Sinnet). Et al., 1990, Genomics 7:331-334). In the plant aspect, a specific primer is designed for the first type of inverted jumper sequence, and a polymerase chain reaction is performed to obtain a molecular marker, which is called Inter-Retrotransposon Amplified Polymorphism (IRAP). (Kalendar et al., 1999, Theor Appl Genet 98: 704-711), or in conjunction with AFLP and SSR, referred to as REMAP (Kalendar et al., 1999, Theor Appl Genet 98:704-711). In addition, the molecular marker for designing a specific primer for the polymerase chain reaction of the MITE sequence in the second type of jumper is called inter-MITE polymorphism (IMP) (Chang et al., 2001, Theor Appl Genet 102:773). -781; WO 00/60113), or with SSR, AFLP (Tang et al., 1995, Genome 6:345-34). US 20050048481 A1 discloses a technique similar to molecular markers for designing polymerase chain reactions with specific primers in the sequence of MITE or SINE.

The invention adopts the polymorphism of the interval of two different jumpers as a molecular marker, and can optimally match different plant species to obtain more molecular markers, and further improve the efficiency of detecting the plant genome.

The present invention is mainly to provide a method for establishing a molecular marker for detecting a polymorphism of a plant genome, which comprises amplifying a genome by a polymerase chain reaction using a primer specific for a first jumper and a second jumper, respectively, to obtain a genome. a segment comprising any sequence between the first hop and any second hop, a segment of the genome comprising any two sequences between the first hops, and/or a segment of the genome comprising any two sequences between the second hops, wherein The first jumper is not identical to the second jumper; and the molecular signature is established by analyzing the polymorphism of the fragments.

The present invention further provides a molecular marker for detecting polymorphism of a plant genome, which is established by the aforementioned method.

Molecular markers are important techniques in molecular biology, especially for cultivar identification, phylogenetic identification or tracking of genetic traits. The present invention utilizes the spacers between different hoppings as molecular markers, and the types of hopping can be selected according to requirements. And obtain the most suitable polymorphism as a molecular marker.

The present invention is mainly to provide a method for establishing a molecular marker for detecting a polymorphism of a plant genome, which comprises amplifying a genome by a polymerase chain reaction using a primer specific for a first jumper and a second jumper, respectively, to obtain a genome. a segment comprising any sequence between the first hop and any second hop, a segment of the genome comprising any two sequences between the first hops, and/or a segment of the genome comprising any two sequences between the second hops, wherein The first jumper is not identical to the second jumper; and the molecular signature is established by analyzing the polymorphism of the fragments.

As used herein, a "jumper" is also referred to as a transposon, a transposer or a metastasis, and refers to a sequence having a transferable positional property. Due to the wide variety of jumpers, their structural characteristics are also different. In general, but not for all jumpers, there are two possibilities for the sequence at both ends: one is the inverted repeat, the two strands are identical by the sequence of 5'→3'; the other is direct The terminal repeat region of the direct repeat has the same sequence at both ends. On the other hand, the internal sequence of the jumper usually contains transgenes related to the transfer position, such as transposase, which controls the translocation reaction, but there are also many internal sequences of the jumper that do not contain transcodeable Fragment. Also, since the jumper may be the same or different from its original direction after being inserted into the new position, the internal sequence of the same jumper may have two different directions.

According to the present invention, the types of the first hop and the second hop are not limited, and may be the same type or different types of hops, but the two may be different. For example, the first hop and the second hop in the present invention may be three different aspects of the invention as follows:

(1) the first hop is a reverse hop and the second hop is an index hop, or an inverse combination thereof;

(2) the first hop and the second hop are all inverted hops, but the two hops are not the same;

(3) The first jumper and the second jumper are all index jumpers, but the two jumpers are not the same.

According to the present invention, the inverted bit hopping means that the intermediate in the case of indexing is a jumper of RNA. Preferably, the inverted bit hopping is selected from the group consisting of a long terminal repeated inverted bit hop and a non-long terminal repeated inverted hop. For example, the long terminal repeated inversion bit jumper may be a Ty1/class copia inversion bit jumper or a Ty3/class gypsy reverse bit jumper; and the non-long terminal repeat inversion bit jumper may be long Disperse nucleons or short-dispersed nucleons.

According to the present invention, an index jumper refers to an intermediate in which the intermediate is a jumper of DNA. Preferably, the index jumper is selected from the group consisting of a derotator, a terminal inverted repeat jumper, and a miniature inverted jumper. For example, the end inverted repeat jumper can be Tc1/mariner, hAT, CACTA, a class-like Mutator, or PIF/Pong.

The term "genome" as used in the present invention, also referred to as a genome, refers to the total amount of genetic material in a cell.

The "polymerase chain reaction" as used in the present invention generally comprises four steps: (1) denaturation of a template to form two single strands; (2) reciprocating the two primers with the two strands of step (1), respectively (3) extending the primers by DNA polymerase; and (4) obtaining the DNA of the two double strands. The above steps are repeated, and amplification is obtained for a specific DNA fragment. The design of the primers for a particular sequence is well known to those of ordinary skill in the art to which the invention pertains, and many commercial softwares are available (Arnheim and Erlich, 1992, Annu. Rev. Biochem. 61: 131-156).

In accordance with the present invention, primers specific for the first and second hops in a polymerase chain reaction can comprise a variety of aspects. For example, the primers can be:

(1) design a forward primer for any fragment of the first jumper and a reverse primer for any fragment of the second jumper, and the primers can be amplified by the polymerase chain reaction to obtain the two genomes in the genome. a segment of any sequence between the first jumper and any second jumper;

(2) designing a forward reference for each of the first hop and the second hop; or designing a reverse priming for each of the first hop and the second hop, the primers are performed A polymerase chain reaction can amplify a fragment comprising a sequence of any first jumper and any second jumper in two different directions in the genome;

(3) designing a forward primer for the end inverted repeat region of the first jumper or the second jumper, or designing a reverse primer for the end jump repeat region of the first jumper or the second jumper, then the primer is Performing a polymerase chain reaction to amplify a fragment containing a sequence of any two first hops in the genome or a sequence of any two second hops in the genome, and the two first hops or the The direction of any two second jumpers may be the same or different; and

(4) designing a forward primer for the end repeat region or the inner sequence region of the first jumper or the second jumper, or designing the end repeat region or the inner sequence region for the first jumper or the second jumper a reverse primer, wherein the primer can be amplified by a polymerase chain reaction to obtain a fragment comprising a sequence of first jumps in any two directions in the genome or a sequence of a second jumper having any two directions different in the genome. Fragment.

In a preferred embodiment of the present invention, the primer is specific to the non-coding element of the first hop and/or the second hop. The non-transcodeable segment may be a specific segment of the jumper, such as an end repeat region of the long segment or an end inverted repeat region; the non-transcodeable segment may also be an inner region of the jumper.

In another preferred embodiment of the present invention, the primer is specific to a transcoding segment of the first hop and/or the second hop. The transcoded fragment can be, for example, a fragment encoding a reverse transcriptase or an integrase.

As used herein, "polymorphism" refers to the variability of certain traits or patterns between the general individuals of the same variety or species. Due to the difference in the number or position of jumps in the evolution process, the polymorphism is caused. Since the jumper is distributed throughout the genome of the plant, the interval between the two jumpers has the opportunity to be amplified by the polymerase chain reaction. Different genomes can produce polytypes due to the different positions of the jumpers or the different lengths of the spacers. a product of sex, the present invention may comprise a fragment comprising any first jumper and any second jumper sequence in the genome, a fragment comprising any two first jumper sequences in the genome, and/or a genome comprising Genomic polymorphism analysis of different types of polymerase chain reaction products of fragments of any two second jumper sequences.

In a specific embodiment of the present invention, the genome includes a segment of any first hop and any second hop sequence, a segment of the genomic sequence comprising any two first hop sequences, and/or a genomic inclusion The polymorphism of the fragments of the two second jumper sequences is analyzed by the size of the amplified fragment, and a molecular marker for detecting the polymorphism of the plant genome is established. The size of the amplified fragment is preferably from about 50 bp to about 4000 bp, more preferably from about 100 bp to about 3500 bp, and more preferably from about 100 bp to about 2000 bp, depending on the ease of experimental manipulation and the conditions of the polymerase chain reaction.

In another embodiment of the present invention, the genome includes a fragment of any first hop and any second hop inter-sequence, a fragment comprising any two first hop sequences in the genome, and/or a genomic inclusion The polymorphism of the fragments of any two second hop sequences is analyzed by the number of amplified fragments, and a molecular marker for detecting the polymorphism of the plant genome is established. The number of amplified fragments is preferably from 1 to about 30; more preferably from 1 to about 20; more preferably from 1 to about 10, in view of the ease of experimental operation and the conditions of the polymerase chain reaction.

The method according to the invention may also be combined with other molecular markers to provide a more detailed polymorphic molecular marker. In a preferred embodiment of the invention, the method further comprises the length of the amplified fragment using restriction enzyme hydrolysis techniques. Polymorphism (AFLP) to establish the molecular marker; in another preferred embodiment of the invention, the method further comprises analyzing the polymorphism of a simple sequence repeat (SSR) to establish the molecular marker. Embodiments of amplified fragment length polymorphism or simple sequence repeats are set forth in Vos et al, 1995, Nucleic Acids Res. 23: 4407-4414 and Tautz, 1989, Nucleic Acids Res. 17:6463-6471, which is It is well known to those of ordinary skill in the art to which the present invention pertains.

According to the method of the present invention, the fragment amplified by the polymerase chain reaction is preferably analyzed for its polymorphism by electrophoresis. For example, the polymorphism is analyzed using agarose gel electrophoresis, acrylamide gel electrophoresis or fluorescent-capillary electrophoresis. The methods of electrophoretic analysis are well known to those of ordinary skill in the art to which the present invention pertains (Chen and Zhou, 1987, Proceedings of Electrophoresis Separation Technology Symposium (ninth), pp. 87-91).

The method of the present invention is suitable for analyzing the pluripotency of various plants. In a preferred embodiment of the invention, the plant is rice or moth orchid.

The method according to the invention establishes molecular markers by using different types of jumpers in different plant genomes and corresponding sets of sets. Because the first jumper is different from the second jumper, the two primers of the polymerase chain reaction can be more diverse through the arrangement and combination, so that the best match can be obtained for different plant species. The most suitable molecular marker. Initially, the primers can be randomly arranged in a small number of samples, and the polymerase chain reaction can be used for screening, and the primer pairing which can amplify the appropriate molecular marker is selected, and then a large number of samples are analyzed.

The present invention further provides a molecular marker for detecting polymorphism of a plant genome, which is established by the aforementioned method.

The molecular markers according to the present invention can be widely applied to various aspects, for example, to identify species or varieties of plants, to identify plant phylogenetic relationships, or to track genetic traits.

The invention is illustrated by the following examples, which are not intended to be limited to the scope of the invention.

Instance General experimental procedure DNA quantification

5 μl of the total DNA extract was dissolved in 495 μl of water and injected into a quartz colorimetric tube. After thorough mixing, the colorimetric tube was placed in a spectrophotometer (Hitachi U-2001 Spectrophotometer), and the absorbance was measured by ultraviolet light at a wavelength of 260 nm. The obtained absorbance value is multiplied by 50, and multiplied by the dilution factor to obtain the DNA concentration (ng/μl) of the original extract. Each DNA sample was diluted to 8 ng/μl according to the concentration of the DNA extract of the different samples, and used as a template DNA for the PCR reaction.

Polymerase chain reaction

The reaction contents and concentrations were as follows: 1.25 units of SuperTherm Gold DNA Polymerase (JMR), 25 mM N-Tris (hydroxymethyl)methyl-3-aminopropanesulfonic acid, 50 mM KCl, 2 mM MgCl ₂ , 1 mM β-mercaptoethanol, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dCTP, 0.2 mM dTTP and 0.4 μM of each primer, finally add 16 ng of template DNA, add 25 μl of total volume of sterile water, place in a 0.2 ml microcentrifuge tube, and add to the surface of the solution. 25 μl of mineral oil to prevent evapotranspiration during thermal cycling.

Electrophoresis analysis

Take 1.5 g of agarose (agarose), heat and dissolve in 100 ml of TBE buffer (40 mM Tris-boric acid, 0.1 mM EDTA, pH 8.0), prepare 1.5% agarose gel, pour into the appropriate template rack, and place the tooth comb ( Comb), made into agarose plate electrophoresis tablets, after coagulation, remove the tooth comb, place the electrophoresis chip into the electrophoresis tank containing TBE buffer, and mix the DNA sample to be separated with 1/10 volume of trace dye. (tracking dye) [0.25% bromophenol blue (bromophenol blue), 40% (w/v) sucrose in water], after the proximal end of the proximal perforation is energized, the DNA sample will run to the positive electrode. The voltage depends on the size of the gel (5V/cm). When the tracer reaches 4/5 of the length of the gel, turn off the power, take out the colloid, and dye it with ethidium bromide (0.5μg/ml). Observe and photograph under UV light.

Example 1

A sample of 16 commercial varieties of Phalaenopsis was used to extract the DNA from the leaves of the samples using the DNeasy Plant Mini Kit (QIAGEN, Germany). Using the extracted DNA as a template, the Ty1/class copia belonging to the long terminal repeating inversion jumper (introduction code: Osr-10-F, 5'-tgg atg gct tgt ctt cca ctt c-3', SEQ ID NO. 1) and the sequence of the MITE jumper belonging to the translocation jumper (introduction code: Mite-stow-os-46, 5'-tca cat cca tcc aaa atc cct-3', SEQ ID NO. 2) The polymerase chain reaction was carried out based on the specific primer set. The reaction conditions in each stage were: DNA denaturation was performed at 94 ° C for 3 minutes and 30 seconds; then the following process was repeated 45 times: 94 ° C denaturation for 45 seconds, 51 ° C for 45 seconds, 72 The extension was extended for 1 minute and 30 seconds at °C; and finally amplified at 72 °C for 5 minutes. The polymerase chain reaction product was separated by 1.5% agarose gel electrophoresis. The results show that different Phalaenopsis varieties can obtain their unique molecular markers (as shown in Figure 1).

Example 2

A sample of 16 commercial varieties of Phalaenopsis was used to extract the DNA from the leaves of the samples using the DNeasy Plant Mini Kit (QIAGEN, Germany). Using the extracted DNA as a template, Tc1/mariner (introduction code: Osmar29i, 5'-ctc cct cca tac cca caa aac a-3', SEQ ID NO. 3) belonging to the translocation jumper and belonging to the long terminal A specific primer set based on the sequence of the Ty3/Gypsy (introduction code: Osr-37-R, 5'-aga ggt gta tcg atc gct aag-3', SEQ ID NO. 4) of the inverted inversion jumper The polymerase chain reaction was carried out. The reaction conditions in each stage were: DNA denaturation was carried out at 94 ° C for 3 minutes and 30 seconds; then the following process was repeated 45 times: denaturation at 94 ° C for 45 seconds, 51 ° C for 45 seconds, and 72 ° C for 1 minute. 30 seconds; and finally amplified at 72 ° C for 5 minutes. The polymerase chain product was separated by 1.5% agarose gel electrophoresis. The results show that different Phalaenopsis varieties can obtain their unique molecular markers (as shown in Figure 2).

Example 3

Taking 22 rice commercial varieties as samples, the leaves of the samples were extracted with DNeasy Plant Mini Kit (QIAGEN, Germany). Using the extracted DNA as a template, Ping/Pong/SNOOPY-like of the PIF/Pong family belonging to the translocation jumper (introduction code: Os-Ping/pong, 5'-aac acc cat tgt gac tgg cc-3) ', SEQ ID NO. 5) and MITE (introduction code: Os-mite-stow-oss, 5'-atg tgg gaa atr cta gaa tga c-3', SEQ ID NO. 6) belonging to the index jumper The sequence-based specific primer set was subjected to polymerase chain reaction. The reaction conditions in each stage were: DNA denaturation was performed at 94 ° C for 3 minutes and 30 seconds; then the following process was repeated 45 times: denaturation at 94 ° C for 45 seconds, and 51 ° C for 45 seconds. The extension was extended at 72 ° C for 1 minute and 30 seconds; and finally at 72 ° C for 5 minutes. The polymerase chain reaction product was separated by 1.5% agarose gel electrophoresis. The results show that different rice varieties can obtain their unique molecular markers (as shown in Figure 3).

Example 4

Taking 22 rice commercial varieties as samples, the leaves of the samples were extracted with DNeasy Plant Mini Kit (QIAGEN, Germany). Using the extracted DNA as a template, Rim2/Hipa belonging to the CACTA family in the translocation jumper (introduction code: Os-Rim2/Hipa-1, 5'-agc gat ctt tag tcc cgg at-3', SEQ ID NO. 7) and the specific primer set based on the sequence of MITE (introduction code: Os-mite-stow-os1, SEQ ID NO. 6) belonging to the translocation jumper is subjected to polymerase chain reaction, and the reaction conditions in each stage are: The DNA denaturation was completed at 94 ° C for 3 minutes and 30 seconds; then the following procedure was repeated 45 times: denaturation at 94 ° C for 45 seconds, 51 ° C for 45 seconds, 72 ° C for 1 minute and 30 seconds; and finally at 72 ° C for 5 minutes . The polymerase chain reaction product was separated by 1.5% agarose gel electrophoresis. The results showed that different varieties of Phalaenopsis can obtain their unique molecular markers (as shown in Figure 4).

The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Therefore, those skilled in the art can make modifications and changes to the above embodiments without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.

Figure 1 shows a sample of 16 Phalaenopsis species, Ty1/category copia (introduction code: Osr-10-F) belonging to the long terminal repeat inversion jumper and MITE belonging to the transposition jumper (introduction code: Mite The specific primers based on the sequence of -stow-os-46) were subjected to polymorphic fragment analysis results. In the figure, the code M molecular DNA reference marker, code 1 to 16, respectively, represents 16 different Phalaenopsis species.

Figure 2 shows the Tc1/mariner (introduction code: Osmar29i) belonging to the transposition jumper and the Ty3/class gypsy belonging to the long terminal repeat inversion jumper for the 16 Phalaenopsis species samples (introduction code: Osr-37) The specific primer set based on the sequence of -R) was subjected to polymorphic fragment analysis. In the figure, the code M molecular DNA reference marker, code 1 to 16, respectively, represents 16 different Phalaenopsis species.

Figure 3 shows the Ping/Pong/SNOOPY-like (introduction code: Os-Ping/pong) of the PIF/Pong family belonging to the transposition jumper and the MITE (introduction code) belonging to the transposition jumper for the samples of 22 rice varieties. The sequence of the polymorphic fragment analysis was performed on a specific primer set based on the sequence of Os-mite-stow-os1). In the figure, the code M molecular DNA reference marker, code 1 to 22, respectively represent 22 different rice varieties.

Figure 4 shows a sample of 22 rice cultivars with Rim2/Hipa (introduction code: Os-Rim2/Hipa-1) belonging to the CACTA family in the transposable jumper and MITE belonging to another translocation jumper (introduction code: Os) The specific primer set based on the sequence of -mite-stow-os1) was subjected to polymorphic fragment analysis. In the figure, the code M molecular DNA reference marker, code 1 to 22, respectively represent 22 different rice varieties.

(no component symbol description)

Claims (36)

A primer pair for detecting the polymorphism of Phalaenopsis genome, comprising a forward primer and a reverse primer, wherein the forward primer is selected from the group consisting of SEQ ID NOs. 1 and 3, and the reverse primer The group consisting of SEQ ID NOs. 2 and 4 is selected. According to the primer pair of claim 1, wherein the forward primer is SEQ ID NOs. 1 and the reverse primer is SEQ ID NO. According to the primer pair of claim 1, wherein the forward primer is SEQ ID NOs. 3 and the reverse primer is SEQ ID NO. A method for detecting a molecular marker of the polymorphism of the Phalaenopsis genome, comprising using a primer pair according to any one of claims 1 to 3 to amplify a Phalaenopsis genome by a polymerase chain reaction to obtain an amplified fragment of the Phalaenopsis genome And establishing the molecular marker by analyzing the polymorphism of the amplified fragment. The method of claim 4, wherein the polymorphism of the amplified fragment is analyzed for the amplified fragment size. The method of claim 5, wherein the amplified fragment size is from about 50 bp to about 4000 bp. The method of claim 6, wherein the amplified fragment size is from about 100 bp to about 3500 bp. The method of claim 7, wherein the amplified fragment size is from about 100 bp to about 2000 bp. The method of claim 4, wherein the polymorphism of the amplified fragment is analyzed for the number of amplified fragments. The method of claim 9, wherein the number of the amplified fragments is from 1 to about 30. The method of claim 10, wherein the number of the amplified fragments is from 1 to about 20. The method of claim 11, wherein the number of the amplified fragments is from 1 to about 10. According to the method of claim 4, which further comprises analyzing the amplified fragment length polymorphisms (AFLP) by restriction enzyme hydrolysis to establish the molecular marker. According to the method of claim 4, it further comprises analyzing the polytype of a simple sequence repeat (SSR) to establish the molecular marker. According to the method of claim 4, it is analyzed by electrophoresis for its polymorphism. A molecular marker for detecting the polymorphism of the Phalaenopsis genome, which is established by the method of any one of claims 4 to 15. According to the molecular marker of claim 16, it is used to identify the species of Phalaenopsis. According to the molecular marker of claim 16, it is used to identify the phylogenetic relationship of Phalaenopsis. According to the molecular marker of claim 16, it is used to track the genetic traits of Phalaenopsis. A primer pair for detecting rice genomic polymorphism, comprising a forward primer and a reverse primer, wherein the forward primer is selected from the group consisting of SEQ ID NOs. 5 and 7, and the reverse primer is SEQ ID NO.6. A method for detecting a molecular marker of rice genomic polymorphism, comprising using a primer pair according to claim 20 to amplify a rice genome by a polymerase chain reaction to obtain an amplified fragment of a rice genome, and analyzing the amplification The polymorphism of the fragment to establish the molecular signature. The method according to claim 21, wherein the polymorphism of the amplified fragment is analyzed for the amplified fragment size. The method of claim 22, wherein the amplified fragment size is from about 50 bp to about 4000 bp. The method of claim 23, wherein the amplified fragment size is from about 100 bp to about 3500 bp. The method of claim 24, wherein the amplified fragment size is from about 100 bp to about 2000 bp. The method according to claim 21, wherein the polymorphism of the amplified fragment is analyzed for the number of amplified fragments. The method of claim 26, wherein the number of amplified fragments is from 1 to about 30. The method of claim 27, wherein the number of amplified fragments is from 1 to about 20. The method of claim 28, wherein the number of amplified fragments is from 1 to about 10. According to the method of claim 21, which further comprises analyzing the amplified fragment length polymorphism by a restriction enzyme hydrolysis method to establish the molecular marker. According to the method of claim 21, it further comprises analyzing the polymorphism of the simple sequence repeat to establish the molecular marker. According to the method of claim 21, it is analyzed by electrophoresis for its polymorphism. A molecular marker for detecting polymorphism of rice genomes, which is established by the method of any one of claims 21 to 32. According to the molecular marker of claim 33, it is used to identify rice varieties. According to the molecular marker of claim 33, it is used to identify the genetic relationship of rice. According to the molecular marker of claim 33, it is used to track the genetic traits of rice.