WO2008117860A1 - 野生種のバラにおける園芸種のバラとの交雑の有無を検定する方法 - Google Patents
野生種のバラにおける園芸種のバラとの交雑の有無を検定する方法 Download PDFInfo
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- WO2008117860A1 WO2008117860A1 PCT/JP2008/056000 JP2008056000W WO2008117860A1 WO 2008117860 A1 WO2008117860 A1 WO 2008117860A1 JP 2008056000 W JP2008056000 W JP 2008056000W WO 2008117860 A1 WO2008117860 A1 WO 2008117860A1
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- rose
- roses
- wild
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- transposon
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to a method for detecting the presence or absence of crossing of a wild-type rose with a garden-type rose.
- the KSN gene which serves as an indicator gene in the present invention, is a gene related to the four-season blooming property of roses obtained from Rosa chinens is spon tanea. This is the result of the insertion of an approximately 9 kb transposon into the rose KSN gene. It has been reported that the insertion of a transposon suppresses the expression of the gene and releases the suppression of flower bud formation at the shoot apex, thus promoting flower bud formation and flowering in all seasons (Iwata et al., Published number) 2006-149202).
- the horticultural rose is homologous to the gene with the transposon inserted. Has already been investigated. On the other hand, wild roses, as a rule, have a homologous KSN gene that does not contain a transposon.
- Rosa chinensis is one of the wild species that became the ancestor of rose horticultural varieties, and it blooms in one season, and the mouth of rosacea chinensis is spon t anea Is a mutant line of this, blooming in all four seasons.
- Patent Document 1 Japanese Unexamined Patent Publication No. 2006-149202 Disclosure of Invention
- wild-type roses can use roses of transgenic horticultural species as pollen parents.
- the present invention provides and uses a method that can easily test whether or not a wild-type rose as a subject is crossed with pollen of a transgenic horticultural species.
- the present inventors have conceived that in wild roses, the KSN gene having a transposon is detected only when crossed with a rose of horticultural species.
- the present invention has been completed by finding that the above problems can be achieved by using the KSN gene as an index. Therefore, the present invention examines whether or not a KSN gene having a transposon is present in the test subject rose in a method for testing whether or not a wild species subject rose is crossed with a garden species rose. If the KSN gene is present, it provides a method characterized in that it is determined that the subject rose is hybridized with a horticultural rose.
- the problem is that the pollen of the horticultural species scatters and crosses with the surrounding wild species. Therefore, in the method of the present invention, typically, the mother is a wild-type rose and the father is a garden-type rose.
- the rose of the horticultural species is a genetically modified rose into which a gene is introduced, and examples of the gene in this case include a gene related to color, for example, a flavonoid derived from a violet family Pansy. 3 ', 5' are monohydroxylase genes.
- Figure 1 shows the results of KSN gene detection by PCR.
- the positions of the specific amplification products of KSN gene and GAPDH gene (endogenous control) including introns detected by PCR are indicated by arrows.
- roses of horticultural species are tetraploid (4x) and bloom in four seasons.
- wild roses are usually diploid (2x) and bloom in one season.
- Horticulture roses are classified as hybrid teas, floripandas, mini roses, etc., but all usually have a homologous KSN gene with a transposon inserted. That is, the rose of the horticultural species referred to in the present invention It means a rose that has a homologous KSN gene with a transposon inserted.
- wild roses that grow naturally in white books include: Neubara, Teri Hanoi rose, Hermanus, ⁇ Evening cane rose, Rough toy rose, ⁇ ⁇ There are, for example, salamander roses, evening cane roses, crush roses, molly roses, fujii roses, scabbard, scented roses, or shrimp roses, which usually have homozygous KSN genes without transposon inserted. That is, the wild-type rose referred to in the present invention means a rose having a KSN gene with no transposon inserted.
- the indicator gene is
- the indicator gene is always present in horticultural roses and not in native wild roses.
- the roses of horticultural species are tetraploid (4x), and the gene that is the index of hybridization must be present in at least 3 of the 4 homologous chromosomes. It is preferably present in all.
- the horticultural roses are tetraploid (4x), while the native wild roses are diploid (2x).
- the gametes arising from horticultural roses are usually 2x, and in the case of wild roses, X, and the hybrid resulting from crossing these is triploid (3x). Therefore, it is necessary that at least three of the homologous genes for roses in horticultural species exist as indicator genes for hybridization.
- the indicator gene exists on all four homologous chromosomes for a reliable test.
- the same sequence as the partial sequence of the base sequence of the indicator gene does not exist in the other part of the chromosome.
- an indicator gene In order to test whether or not an indicator gene is present in a test plant, it is usually necessary to amplify the gene. For this purpose, a pair of primers complementary to the target gene is used. The PCR method is convenient. In this case, if the sequence to be hybridized with the primer is present in addition to the target gene, a result can be obtained that can be determined to be present even if the original target gene does not exist. This requirement is therefore important in the context of primer design.
- a gene that satisfies the above requirements is the KSN gene with a transposon inserted.
- the KSN gene (Publication 2006-149202), which is reported as a gene related to the blooming of the four seasons of roses, exists as a complete gene that codes for 519 amino acids in wild species, but about 9 It is clear that a kb transposon has been inserted. In other words, since this transposon does not exist in wild species but always exists in horticultural species, the KSN gene containing this transposon satisfies the requirement (1).
- the KSN gene containing this transposon is present in all four horticultural rose genes. Therefore, this gene satisfies the above requirement (2).
- the KSN gene containing this transposon also satisfies the above requirement (3) by selecting a primer.
- the KSN gene (SEQ ID NO: 1) and the base sequence of the transposon inserted therein (SEQ ID NO: 2) are already known.
- the KSN gene with this transposon inserted is only found in horticultural roses. It exists differently.
- one plasmid having a sequence identical or substantially complementary to the sequence shown in SEQ ID NO: 1, and the other shown in SEQ ID NO: 2 It is necessary to use a pair of primers having the same or substantially complementary sequence as the sequence.
- Each primer is an oligonucleotide having a size of 10 bases or more, preferably 15 bases or more, and 50 bases or less, preferably 30 bases or less. is there.
- the region to be amplified in the transposon is not particularly limited, but it is not preferable that both primer pairs hybridize with a portion other than the transposon.
- a primer pair that satisfies such requirements is a forward primer: CATA TTATGGCATAGGGTGTGGC (SEQ ID NO: 3) and a reverse primer TGTAATCTGTAGGAGATCCCATGC (SEQ ID NO: 4).
- Reference example 1 Acquisition of horticultural species-specific genes of roses by RAPD (Random Amplified Polymorphic DNA) analysis
- the reaction was carried out at 94 for 5 minutes, followed by 25 cycles of 2 minutes at 94, 1 minute, 55, 1 minute and 72, and finally at 72 for 7 minutes.
- the obtained PCR product was subjected to 0.8% agarose gel electrophoresis, and the migration patterns between wild species and horticultural species were compared.
- a band specifically detected only in horticultural species was excised from an agarose gel and purified by GENECLEAN Turbo Kit (Funakoshi Co., Ltd.) according to the method recommended by the manufacturer. Finally, these were subcloned into pCR2.1 TOPO vector (INVITROGEN) by the method recommended by the manufacturer, and the sequence was determined by sequence analysis.
- each amplified fragment was analyzed.
- a 20-mer oligo primer containing a common primer sequence at both ends of each fragment was prepared, and a combination of oligo primers that specifically detected an amplification product only in horticultural species (Labande, WKS82) was examined.
- a PCR reaction solution comprising 2 M of each oligoprimer, 1 xdNTP mixture, and Ex Taa Buffer 0.05 M was prepared using 10 ng of each genomic DNA of Miyakoibara, Labande, and WKS82 as a saddle type. The reaction was carried out at 94 for 5 minutes, then 94 at 1 minute, 55 at 1 minute, 72 at 2 minutes for 25 minutes, and finally at 72 at 7 minutes.
- the obtained PCR product was subjected to 0.8% agarose gel electrophoresis, and a combination of oligo primers capable of specifically detecting an amplification product only in horticultural species was examined.
- a combination of oligo primers considered to give a broad band was selected, and the experiment shown in Reference Example 2 was conducted.
- the RAPD markers obtained this time are not included in all four homologous chromosomes, and some of the four homologous chromosomes in horticultural species It was inferred that it was derived from the gene sequence present only above, that is, it was judged to be inappropriate as an indicator of the presence or absence of hybridization.
- Example 1 Determination of the presence or absence of crosses in progeny using the detection of the KSN gene as an indicator
- KSN gene containing transposon which is known to be specifically detected in roses having four seasons blooming properties, when wild roses are pollinated with horticultural rose pollen (W02004 / 070036) Detection The presence or absence of hybridization was tested using as an index.
- the reaction was carried out at 94 for 5 minutes, then at 94 for 1 minute, 55 ", 1 minute, 72" C, 2 minutes, 25 cycles, and finally at 72 for 7 minutes.
- the obtained PCR product was subjected to 0.8% agarose gel electrophoresis to confirm the presence or absence of a specific band of about 1.2 kb.
- the form of the individual in which the band was detected showed an intermediate character between the horticultural species and the wild species.
- the KSN gene containing transposon was present in the individuals who were able to do so. From this, it was found that individuals who do not have a KSN gene containing a transposon can be judged not to be hybrids between horticultural paras and wild roses.
- Example 2 Determination of the presence of KSN gene in wild rose
- the presence or absence of a KSN gene containing a transposon in each wild-type rose was evaluated.
- Example 1 The presence or absence of a KSN gene (W02004 / 070036) containing a transposon was tested according to the method described above.
- the method of the present invention using the KSN gene containing a transposon as an index can suitably use wild species of rose as a test subject.
- Genomic DNA was extracted from the recovered seeds using Nucleon PHYTOPUEE for PLANT DNA EXTRACTION KIT (Amershai Biosciences) according to the method recommended by the manufacturer. After amplification with the REPLI-g Midi Kit (QIAGEN), PCR analysis using the presence or absence of a transposon-inserted KSN gene as an indicator indicates the presence or absence of hybridization with the host or recombinant, and transgene transfer. The presence or absence of was analyzed. As an endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used.
- GPDH glyceraldehyde-3-phosphate dehydrogenase
- Rh GAPDH-237F 5'-TGT CAT CTC TGC CCC AAG TAA GG— 3 '(SEQ ID NO: 5)
- Rh GAPDH-724R 5 '-CAA CAT CCT CAT CGG TGT AAC CC— 3' (sequence number: 6)
- the results are shown in Tables 1 and 2.
- the fruiting rate was extremely low whether the host or the recombinant plant was the pollen parent.
- the obtained seedlings were analyzed by PCR, but a KSN gene containing a transposon was detected.
- no transgene derived from the recombinant was detected.
- the seeds that did not germinate were collected again and observed for seed enrichment, most of the seeds were "Shi (no seed contents)" and normal embryos were confirmed. There were very few individuals. These were similarly analyzed by the PCR method.
- the assay method of the present invention it was possible to evaluate the presence or absence of a cross between a wild species rose and a garden species rose very simply.
- a transgene is not transmitted to a progeny, for example, because the transgene is not contained in a recombinant pollen cell, it is often difficult to analyze the cross.
- the detection method of the present invention is used, the presence or absence of hybridization can be easily determined. Therefore, the test method of the present invention first determines the presence or absence of crossing, and if it is determined that crossing, the transgene is further determined by examining the presence or absence of transgene transmission. It is possible to infer whether or not it exists in germ cells. Table 1. Fruiting rates with wild species (Neubara, Terihanoba rose, Hermanus) by artificial mating and transgene detection rate in germinating individuals
- the presence or absence of a cross between a wild-type rose and a garden-type rose under natural conditions was evaluated.
- the assay method of the present invention it was possible to evaluate the presence or absence of a cross between a wild species rose and a garden species rose very simply.
- a transgene is not transmitted to a progeny, for example, because the transgene is not contained in a recombinant pollen cell, it is often difficult to analyze the cross.
- the presence or absence of hybridization can be easily determined by the detection method of the present invention, introduction by hybridization It was also possible to easily analyze the presence or absence of gene transfer.
- Example 5 Verification of the presence of crosses with horticultural species roses in seeds collected from native wild roses
- the presence or absence of a cross between a wild-type rose and a horticultural rose under natural conditions was evaluated.
- Example 3 According to the method of Example 3, from the seeds of wild roses that grow naturally, the presence or absence of the transposon-inserted KSN gene was used as an index. By CR analysis, the presence or absence of crossing with horticultural roses was verified. In addition, as an endogenous control, the dalycel aldehyde-3-phosphate dehydrogenase (GAPDH) gene was used.
- GPDH dalycel aldehyde-3-phosphate dehydrogenase
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2008230321A AU2008230321B2 (en) | 2007-03-23 | 2008-03-21 | Method for determination of presence of crossing with garden-species rose plant in wild-species rose plant |
EP08722920.9A EP2128274B1 (en) | 2007-03-23 | 2008-03-21 | Method for determination of presence of crossing with garden-species rose plant in wild-species rose plant |
JP2009506377A JPWO2008117860A1 (ja) | 2007-03-23 | 2008-03-21 | 野生種のバラにおける園芸種のバラとの交雑の有無を検定する方法 |
CA002681297A CA2681297A1 (en) | 2007-03-23 | 2008-03-21 | Method for determination of presence of crossing with cultivated rose in wild rose |
US12/532,246 US8206928B2 (en) | 2007-03-23 | 2008-03-21 | Method for determination of presence of crossing with cultivated rose in wild rose |
ES08722920.9T ES2462944T3 (es) | 2007-03-23 | 2008-03-21 | Método para determinar en un rosal silvestre la presencia de cruzamiento con un rosal cultivado |
Applications Claiming Priority (2)
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JP2007-077882 | 2007-03-23 | ||
JP2007077882 | 2007-03-23 |
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WO2008117860A1 true WO2008117860A1 (ja) | 2008-10-02 |
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PCT/JP2008/056000 WO2008117860A1 (ja) | 2007-03-23 | 2008-03-21 | 野生種のバラにおける園芸種のバラとの交雑の有無を検定する方法 |
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Country | Link |
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US (1) | US8206928B2 (ja) |
EP (1) | EP2128274B1 (ja) |
JP (1) | JPWO2008117860A1 (ja) |
KR (1) | KR20090125086A (ja) |
CN (1) | CN101617058A (ja) |
AU (1) | AU2008230321B2 (ja) |
CA (1) | CA2681297A1 (ja) |
CO (1) | CO6141480A2 (ja) |
EC (1) | ECSP099647A (ja) |
ES (1) | ES2462944T3 (ja) |
TW (1) | TW200846476A (ja) |
WO (1) | WO2008117860A1 (ja) |
Cited By (1)
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CN108841992A (zh) * | 2018-07-26 | 2018-11-20 | 安徽中医药大学 | 鉴别何首乌和棱枝何首乌的分子特异性标记引物及方法 |
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CN109258456B (zh) * | 2018-11-06 | 2022-04-08 | 兰州市农业科技研究推广中心 | 一种中国苦水玫瑰人工授粉的方法 |
Citations (4)
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US5480789A (en) | 1991-04-01 | 1996-01-02 | Florigene Europe B.V. | Genetically transformed rose plants and methods for their production |
JPH11512289A (ja) | 1995-09-13 | 1999-10-26 | プラント バイオサイエンス リミティド | 開花遺伝子 |
WO2003097869A2 (de) * | 2002-05-17 | 2003-11-27 | Con / Cipio Gmbh | Mikrosatellitenmarker für genetische analysen und zur unterscheidung von rosen |
WO2004070036A1 (ja) * | 2003-02-04 | 2004-08-19 | Wakunaga Pharmaceutical Co., Ltd. | 被子植物の四季咲き性に関与するタンパク質および遺伝子 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US5948955A (en) * | 1991-07-13 | 1999-09-07 | International Flower Developments Pty Ltd | Transgenic plants having altered anthocyann levels |
JP2004275048A (ja) * | 2003-03-14 | 2004-10-07 | Rikogaku Shinkokai | プラスチッドの数又は大きさを制御するdna及びタンパク質 |
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2008
- 2008-03-21 WO PCT/JP2008/056000 patent/WO2008117860A1/ja active Application Filing
- 2008-03-21 CA CA002681297A patent/CA2681297A1/en not_active Abandoned
- 2008-03-21 KR KR1020097018803A patent/KR20090125086A/ko not_active Application Discontinuation
- 2008-03-21 TW TW097110029A patent/TW200846476A/zh unknown
- 2008-03-21 EP EP08722920.9A patent/EP2128274B1/en not_active Not-in-force
- 2008-03-21 JP JP2009506377A patent/JPWO2008117860A1/ja active Pending
- 2008-03-21 CN CN200880005497A patent/CN101617058A/zh active Pending
- 2008-03-21 ES ES08722920.9T patent/ES2462944T3/es active Active
- 2008-03-21 US US12/532,246 patent/US8206928B2/en not_active Expired - Fee Related
- 2008-03-21 AU AU2008230321A patent/AU2008230321B2/en not_active Expired - Fee Related
-
2009
- 2009-09-23 EC EC2009009647A patent/ECSP099647A/es unknown
- 2009-09-23 CO CO09103864A patent/CO6141480A2/es unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US5480789A (en) | 1991-04-01 | 1996-01-02 | Florigene Europe B.V. | Genetically transformed rose plants and methods for their production |
JPH11512289A (ja) | 1995-09-13 | 1999-10-26 | プラント バイオサイエンス リミティド | 開花遺伝子 |
WO2003097869A2 (de) * | 2002-05-17 | 2003-11-27 | Con / Cipio Gmbh | Mikrosatellitenmarker für genetische analysen und zur unterscheidung von rosen |
WO2004070036A1 (ja) * | 2003-02-04 | 2004-08-19 | Wakunaga Pharmaceutical Co., Ltd. | 被子植物の四季咲き性に関与するタンパク質および遺伝子 |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108841992A (zh) * | 2018-07-26 | 2018-11-20 | 安徽中医药大学 | 鉴别何首乌和棱枝何首乌的分子特异性标记引物及方法 |
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Publication number | Publication date |
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JPWO2008117860A1 (ja) | 2010-07-15 |
AU2008230321A2 (en) | 2009-10-15 |
AU2008230321A1 (en) | 2008-10-02 |
ECSP099647A (es) | 2009-10-30 |
CO6141480A2 (es) | 2010-03-19 |
ES2462944T3 (es) | 2014-05-26 |
KR20090125086A (ko) | 2009-12-03 |
EP2128274B1 (en) | 2014-05-07 |
CA2681297A1 (en) | 2008-10-02 |
EP2128274A4 (en) | 2010-07-07 |
CN101617058A (zh) | 2009-12-30 |
US20100143914A1 (en) | 2010-06-10 |
EP2128274A1 (en) | 2009-12-02 |
US8206928B2 (en) | 2012-06-26 |
TW200846476A (en) | 2008-12-01 |
AU2008230321B2 (en) | 2013-04-04 |
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