CN105331605B - A kind of Gypsy retrotransponsonses of P genome specifics and its application - Google Patents
A kind of Gypsy retrotransponsonses of P genome specifics and its application Download PDFInfo
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Abstract
The invention discloses a kind of Gypsy retrotransponsonses of P genome specifics and its application.The Gypsy retrotransponsonses for the P genome specifics that the present invention is provided are following (a) or (b)：(a) DNA molecular shown in sequence 1 from the nucleotides of 5 ' end the 531st 1575；(b) DNA molecular shown in sequence 1.The present invention also protects the special primer pair that the single strand dna shown in the sequence 2 by sequence table and the single strand dna shown in the sequence 3 of sequence table are constituted.Meaning of the present invention is as follows：(1) evolution of the interspersed repetitive sequence pAcPR1 acquisition for studying Wheatgrass plays an important roll；(2) due to the acquisition of a large amount of wheat wheatgrass translocation lines so that propose new challenge to the chromatinic quick detection of external source wheatgrass.And obtaining for the specific molecular marker based on P genome retrotransponsonses can carry out efficient detection to the wheatgrass chromatin under Wheat Background.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of Gypsy retrotransponsonses of P genome specifics and its should
With.
Background technology
Repetitive dna sequence is the chief component of most plants genome.It can be divided into two classes：Tandem sequence repeats sequence
Row and interspersed repetitive sequence.And most interspersed repetitive sequences are the characteristics of have transposons and retrotransponsons and feature.It is this kind of scattered
Reiterated DNA sequences not still the important component of Plant Genome and also be Plant Genome evolve main power.Long end
End retrotransponsons is the focus of research in repetitive sequence.In recent years, some isolated new scattered repetitions from plant
Sequence, this evolution to be better understood by the structure and Plant Genome of retrotransponsons provides help.And in Agropyron also not
See the research of this respect.There is the interspersed repetitive sequence of retrotransponsons feature so obtaining one and being distributed in the whole chromosomes of wheatgrass
With critically important Evolutionary Significance.
As the important genetic resources of wheat flour quality, Wheatgrass has many high yield characteristics, and such as tillering ability is strong, many
Many small ears etc. are spent, while resistant to the biotic such as abiotic stress and powdery mildew, rust such as drought, cold.The profits such as Li Lihui
Hybridized with the wheatgrass (A.cristatum) for picking up from Xinjiang, China with common wheat (Triticum aestivum L.) Fukuho,
By being further returned multiple wheats-wheatgrass Alien disomic addition lines are obtained with selfing.The success of wheat-wheatgrass distant hybridization
And alien addition line be established as the favorable genes of wheatgrass P genomes are transferred to wheat there is provided premise.A large amount of translocation lines
Obtain and provide possibility for effective utilize of wheatgrass favorable genes, and the chromatinic tracking of wheatgrass and identification are accomplished by one simply
Effective means.Genome specific sequence, the especially acquisition of full-length genome interspersed repetitive sequence and PCR point based on this exploitation
Son mark can be tracked detection as the molecular label of genome specific to the alien chromatin for importing wheat.
In summary, the meaning just because of retrotransponsons in Plant Evolution, and it is important in wheat flour quality
Effect, is badly in need of developing such interspersed repetitive sequence in Agropyron.
The content of the invention
It is an object of the invention to provide a kind of Gypsy retrotransponsonses of P genome specifics and its application.
The Gypsy retrotransponsonses (molecular labeling is named as specific DNA molecular) for the P genome specifics that the present invention is provided, be
As follows (a) or (b)：(a) DNA molecular shown in the sequence 1 of sequence table from the nucleotides of 5 ' end 531-1575；(b) sequence
DNA molecular shown in the sequence 1 of table.
The present invention also protects a species-specific primers pair, as the single strand dna shown in the sequence 2 of sequence table and sequence table
Single strand dna composition shown in sequence 3.
The present invention also protects the special primer to identifying whether vegetable material to be measured contains the heredity from wheatgrass
Application in material.
The present invention also protects whether a kind of detection vegetable material to be measured contains the method for the inhereditary material from wheatgrass, wraps
Include following steps：Using the genomic DNA of vegetable material to be measured as template, using the special primer to entering performing PCR amplification, if
Obtain pcr amplification product, vegetable material to be measured and contain inhereditary material from wheatgrass, if do not obtain pcr amplification product,
Vegetable material to be measured may not contain the inhereditary material from wheatgrass.
The size of the pcr amplification product concretely 1045 ± 5bp.
The present invention also protects the specific DNA molecular identifying whether vegetable material to be measured contains the heredity from wheatgrass
Application in material.
The present invention also protects whether a kind of detection vegetable material to be measured contains the method for the inhereditary material from wheatgrass, wraps
Include following steps：Using the specific DNA molecular as probe, treat measuring plants chromosome and carry out FISH, if
Contain the inhereditary material from wheatgrass with fluorescence signal, vegetable material to be measured, if without fluorescence signal, treating measuring plants
Material may not contain the inhereditary material from wheatgrass.
Treated described in any of the above measuring plants can for wheatgrass Z559, wheatgrass Z1842, wheat-wheatgrass translocation line WAT32, wheat-
Wheatgrass translocation line WAT31, wheatgrass Z1750, wheat China spring, wheat Fukuho, einkorn wheat MO4, durum wheat capital DR3、
Timopheevi wheat TI1, shape of tail goatweed Ae14, aegilops tauschii Y93, Aegilops comosa Y258, Ae.speltoides
Ae49, Aegilops umbelluata Y39, rye RM2161, barley Zhejiang skin 1, haynaldia villosa Z1731, Leymus racemosus R429, false roegneria kamoji
Z1365, roegneria kamoji Z2192, wheat-wheatgrass 6P disomic addition lines 4844-12, wheat-wheatgrass 6P long arm ends system, wheat-ice
Careless 6P galianconism end system, wheat-wheatgrass addition line 5113, wheat-wheatgrass addition line 5114, wheat-wheatgrass addition line II-26,
Wheat-wheatgrass addition line II-29-2i, wheat-wheatgrass addition line 5106, wheat-wheatgrass addition line II-5-1, wheat-wheatgrass are attached
Plus be II-4-2, it is wheat-wheatgrass addition line 5038, wheat-wheatgrass addition line 5043, wheat-wheatgrass addition line II-21-2, small
Wheat-wheatgrass addition line II-21-6, wheat-wheatgrass addition line II-9-3, wheat-wheatgrass addition line II-30-5, wheat-wheatgrass are attached
Plus be II-1-3, wheat-wheatgrass addition line II-3-1, wheat-wheatgrass addition line II-7-1 or wheat-wheatgrass addition line II-8-
1。
The present invention also protects the DNA molecular shown in the sequence 1 of sequence table as the application of retrotransponsons.
Meaning of the present invention is as follows：(1) evolution of the interspersed repetitive sequence pAcPR1 acquisition for research Wheatgrass
Play an important roll；(2) due to the acquisition of a large amount of wheats-wheatgrass translocation line so as to the chromatinic quick detection of external source wheatgrass
Propose new challenge.And the acquisition of the specific molecular marker based on P genome retrotransponsonses can be to the ice under Wheat Background
Careless chromatin carries out efficient detection.
Brief description of the drawings
Fig. 1 is wheat-wheatgrass 6P disomic addition lines 4844-12 FISH results.
Fig. 2 is wheatgrass Z1842 FISH results.
Fig. 3 is wheat-wheatgrass translocation line WAT32 and wheat-wheatgrass translocation line WAT31 FISH results.
Fig. 4 is the electrophoretogram of agarose gel electrophoresis in embodiment 2.
Fig. 5 is the electrophoretogram of agarose gel electrophoresis in embodiment 3.
To entering performing PCR amplification, simultaneously agarose is solidifying to use the special primer that AcPR1F and AcPR1R is constituted in embodiment 4 by Fig. 6
The electrophoretogram of gel electrophoresis.
Fig. 7 is expanded and agar to enter performing PCR using the ControlF and ControlR control primer pairs constituted in embodiment 4
The electrophoretogram of sugared gel electrophoresis.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.
Wheatgrass Z559 (tetraploid, 4n=28)：Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection
With research center (Wu, J., X.Yang, H.Wang, H.Li, L.Li, X.Li, et al.2006.The introgression of
chromosome 6P specifying for increased numbers of florets and kernels from
Agropyron cristatum into wheat.Theoretical and Applied Genetics114:13-20.)。
Wheatgrass Z1842 (diploid, 2n=14, P genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm
Protection of resources and research center.
Wheatgrass Z1750 (hexaploid, 6n=42, P genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm
Protection of resources and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Wheat China spring (common wheat, hexaploid, 6n=42, ABD genomes)：Chinese Academy of Agricultural Sciences's crop science is ground
Study carefully institute's crop germplasm resource protection and research center (Yang Guohui, Yang Xinming, Wang Ruihui, Gao Ainong, Li Lihui, Liu Weihua
.2010. wheat-inhibitory action Science Bulletin of the restructuring P chromosomes of wheatgrass addition line 14 to Ph genes:463-467.).
Wheat Fukuho (common wheat, hexaploid, 6n=42, ABD genomes)：Chinese Academy of Agricultural Sciences's crop science is ground
Study carefully institute's crop germplasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Einkorn wheat MO4(diploid, 2n=14, A genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science makees
Thing plasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Durum wheat capital DR3(tetraploid, 4n=28, AB genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science makees
Thing plasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Timopheevi wheat TI1(tetraploid, 4n=28, AG genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science
Crop germplasm resource is protected and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Shape of tail goatweed Ae14 (diploid, 2n=14, C genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science makees
Thing plasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Aegilops tauschii Y93 (diploid, 2n=14, D genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science's Crop Species
Matter protection of resources and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Aegilops comosa Y258 (diploid, 2n=14, M genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science makees
Thing plasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Ae.speltoides Ae49 (diploid, 2n=14, S genomes)：Chinese Academy of Agricultural Sciences's crop science is ground
Study carefully institute's crop germplasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Aegilops umbelluata Y39 (diploid, 2n=14, U genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science crop
Plasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Rye RM2161 (diploid, 2n=14, R genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science's Crop Species
Matter protection of resources and research center.
Barley Zhejiang skin 1 (diploid, 2n=14, H gene group)：Institute of Crop Science, Chinese Academy of Agricultural Science's Crop Species
Matter protection of resources and research center.
Haynaldia villosa Z1731 (diploid, 2n=14, V genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science's Crop Species
Matter protection of resources and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Leymus racemosus R429 (diploid, 2n=14, Ns genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science's Crop Species
Matter protection of resources and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
False roegneria kamoji Z1365 (diploid, 2n=14, St genomes)：Institute of Crop Science, Chinese Academy of Agricultural Science makees
Thing plasm resource protection and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Roegneria kamoji Z2192 (diploid, 2n=14, Y gene group)：Institute of Crop Science, Chinese Academy of Agricultural Science's Crop Species
Matter protection of resources and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao, X.Zhang, et
al.2010.Cloning and characterization of repetitive sequences and development
of SCAR markers specific for the P genome of Agropyron cristatum.Euphytica
172:363-372.)。
Wheat-wheatgrass 6P disomic addition lines 4844-12 (has+1 pair of wheatgrass 6P chromosome of 42 wheat bar chromosome)：China
The protection of crop science research institute of Academy of Agricultural Sciences crop germplasm resource and research center (Wu, J., X.Yang, H.Wang, H.Li,
L.Li,X.Li,et al.2006.The introgression of chromosome 6P specifying for
increased numbers of florets and kernels from Agropyron cristatum into
wheat.Theoretical and Applied Genetics 114:13-20.)。
Wheat-wheatgrass 6P long arm ends system (has+1 pair of wheatgrass 6P chromosome long arm of 42 chromosomes of wheat)：Chinese agriculture
The crop germplasm resource protection of crop science research institute of the industry academy of sciences and research center.
Wheat-wheatgrass 6P galianconism end system (has+1 pair of wheatgrass 6P the short arm of a chromosome of 42 chromosomes of wheat)：Chinese agriculture
The crop germplasm resource protection of crop science research institute of the industry academy of sciences and research center.
Wheat-wheatgrass addition line 5113 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：The Chinese Academy of Agricultural Sciences
The protection of crop science research institute crop germplasm resource and research center (Han, H., L.Bai, J.Su, J.Zhang, L.Song,
A.Gao,et al.2014.Genetic rearrangements of six Wheat–Agropyron cristatum 6P
addition lines revealed by molecular markers.PloS one 9:e91066.)。
Wheat-wheatgrass addition line 5114 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：The Chinese Academy of Agricultural Sciences
The protection of crop science research institute crop germplasm resource and research center (Han, H., L.Bai, J.Su, J.Zhang, L.Song,
A.Gao,et al.2014.Genetic rearrangements of six Wheat–Agropyron cristatum 6P
addition lines revealed by molecular markers.PloS one 9:e91066.)。
Wheat-wheatgrass addition line II-26 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The protection of crop science research institute of institute crop germplasm resource and research center (Han, H., L.Bai, J.Su, J.Zhang, L.Song,
A.Gao,et al.2014.Genetic rearrangements of six Wheat–Agropyron cristatum 6P
addition lines revealed by molecular markers.PloS one 9:e91066.)。
Wheat-wheatgrass addition line II-29-2i (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Chinese agriculture section
The protection of crop science research institute of institute crop germplasm resource and research center (Han, H., L.Bai, J.Su, J.Zhang,
L.Song,A.Gao,et al.2014.Genetic rearrangements of six Wheat–Agropyron
cristatum 6P addition lines revealed by molecular markers.PloS one 9:
e91066.)。
Wheat-wheatgrass addition line 5106 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：The Chinese Academy of Agricultural Sciences
The protection of crop science research institute crop germplasm resource and research center (Han, H., L.Bai, J.Su, J.Zhang, L.Song,
A.Gao,et al.2014.Genetic rearrangements of six Wheat–Agropyron cristatum 6P
addition lines revealed by molecular markers.PloS one 9:e91066.)。
Wheat-wheatgrass addition line II-5-1 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The protection of crop science research institute of institute crop germplasm resource and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao,
X.Zhang,et al.2010.Cloning and characterization of repetitive sequences and
development of SCAR markers specific for the P genome of Agropyron
cristatum.Euphytica 172:363-372.)。
Wheat-wheatgrass addition line II-4-2 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The protection of crop science research institute of institute crop germplasm resource and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao,
X.Zhang,et al.2010.Cloning and characterization of repetitive sequences and
development of SCAR markers specific for the P genome of Agropyron
cristatum.Euphytica 172:363-372.)。
Wheat-wheatgrass addition line 5038 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：The Chinese Academy of Agricultural Sciences
The protection of crop science research institute crop germplasm resource and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao,
X.Zhang,et al.2010.Cloning and characterization of repetitive sequences and
development of SCAR markers specific for the P genome of Agropyron
cristatum.Euphytica 172:363-372.)。
Wheat-wheatgrass addition line 5043 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：The Chinese Academy of Agricultural Sciences
The crop germplasm resource protection of crop science research institute and research center.
Wheat-wheatgrass addition line II-21-2 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Chinese agriculture section
The protection of crop science research institute of institute crop germplasm resource and research center (Wu, M., J.Zhang, J.Wang, X.Yang,
A.Gao,X.Zhang,et al.2010.Cloning and characterization of repetitive sequences
and development of SCAR markers specific for the P genome of Agropyron
cristatum.Euphytica 172:363-372.)。
Wheat-wheatgrass addition line II-21-6 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Chinese agriculture section
The protection of crop science research institute of institute crop germplasm resource and research center (Wu, M., J.Zhang, J.Wang, X.Yang,
A.Gao,X.Zhang,et al.2010.Cloning and characterization of repetitive sequences
and development of SCAR markers specific for the P genome of Agropyron
cristatum.Euphytica 172:363-372.)。
Wheat-wheatgrass addition line II-9-3 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The protection of crop science research institute of institute crop germplasm resource and research center (Wu, M., J.Zhang, J.Wang, X.Yang, A.Gao,
X.Zhang,et al.2010.Cloning and characterization of repetitive sequences and
development of SCAR markers specific for the P genome of Agropyron
cristatum.Euphytica 172:363-372.)。
Wheat-wheatgrass addition line II-30-5 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Chinese agriculture section
The crop germplasm resource protection of crop science research institute of institute and research center.
Wheat-wheatgrass addition line II-1-3 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The crop germplasm resource protection of crop science research institute of institute and research center.
Wheat-wheatgrass addition line II-3-1 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The crop germplasm resource protection of crop science research institute of institute and research center.
Wheat-wheatgrass addition line II-7-1 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The crop germplasm resource protection of crop science research institute of institute and research center.
Wheat-wheatgrass addition line II-8-1 (has+1 pair of wheatgrass P chromosome of 42 chromosomes of wheat)：Scientia Agricultura Sinica
The crop germplasm resource protection of crop science research institute of institute and research center.
Wheat-wheatgrass translocation line WAT32：Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection is with grinding
Study carefully center (Luan Y, Wang X, Liu W, Li C, Zhang J et al. (2010) Production and
identification of wheat-Agropyron cristatum 6P translocation lines.Planta
232:501-510.doi:10.1007/s00425-010-1187-9.PubMed:20490543.)。
Wheat-wheatgrass translocation line WAT31：Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection is with grinding
Study carefully center (Luan Y, Wang X, Liu W, Li C, Zhang J et al. (2010) Production and
identification of wheat-Agropyron cristatum 6P translocation lines.Planta
232:501-510.doi:10.1007/s00425-010-1187-9.PubMed:20490543.)。
Embodiment 1, pAcPR1 discovery and its distribution characteristics on wheatgrass chromosome
First, pAcPR1 discovery
Using wheat-wheatgrass 6P galianconism end system as material, 6P galianconism has been obtained by the method for micro-dissections and DOP-PCR
DNA sequencing fragment.These sequences are connected to pMD19-T carriers, imports in Escherichia coli, obtains the DNA sequence dna of 6P galianconism
Fragment library.Wheatgrass Z559 is filtered out with dot hybridization and the discrepant DNA clones of wheat Fukuho are sequenced.
Analyzed through BLAST, there is a length is the DNA fragmentation that 1793bp, G/C content are 37.9%, by the DNA fragmentation
PAcPR1 is named as, as shown in sequence 1 in sequence table.
One long end (LTR) Gypsy class retrotransposition subsequence phases in pAcPR1 and wheat 3B- specific B AC storehouses
Like property at most (being 73%), the retrotransposon of the wheat is a cgt0464b part.
PAcPR1 is a new sequence.
2nd, distribution characteristics and specificity verification of the pAcPR1 on wheatgrass chromosome
Using pAcPR1 as probe, respectively to wheat-wheatgrass 6P disomic addition lines 4844-12, wheatgrass Z1842, wheat-wheatgrass
Translocation line WAT-32 and wheat-wheatgrass translocation line WAT-31 carry out FISH (FISH) analyses.
Wheat-wheatgrass 6P disomic addition lines 4844-12 FISH results are shown in Fig. 1.As a result show, pAcPR1 contaminates in whole piece 6P
All it is distributed on colour solid, and the equal no signal on 42 chromosomes in wheat source.
Wheatgrass Z1842 FISH results are shown in Fig. 2.PAcPR1 is distributed on 14 chromosomes, and for every dyeing
PAcPR1 distribution is throughout whole chromosome for body.
Wheat-wheatgrass translocation line WAT-32 and wheat-wheatgrass translocation line WAT-31 FISH results are shown in Fig. 3.Explanation
PAcPR1 can be used for the detection of different type translocation chromosome as probe.
As a result show：PAcPR1 is the distinctive sequence of wheatgrass genome, and pAcPR1 is distributed across all dyeing of wheatgrass
Body, wheatgrass chromosome can be identified by probe of pAcPR1.
The development and application of embodiment 2, special primer pair
1st, based on pAcPR1 sequences Designs special primer to as follows：
AcPR1F (sequence 2 in sense primer, sequence table)：5’-CCCCTTCATTAAGGTATTGTTCC-3’；
AcPR1R (sequence 3 in anti-sense primer, sequence table)：5’-CTTGTCCACATGTTGTGTGCTAT-3’.
2nd, the genomic DNA of each sample to be tested is extracted respectively.
3rd, respectively using each genomic DNA as template, the special primer obtained using step 1 is obtained to entering performing PCR amplification
Pcr amplification product.
4th, each pcr amplification product for obtaining step 3 carries out 1% agarose gel electrophoresis, as a result sees Fig. 4.
In Fig. 4, M correspondences marker, 1 corresponding wheatgrass Z559,2 corresponding wheat Fukuho, 3 corresponding wheats-wheatgrass 6P disomes
Addition line 4844-12,4 corresponding wheats-wheatgrass addition line 5113,5 corresponding wheats-wheatgrass addition line 5114,6 corresponding wheat-ice
Careless addition line II-26,7 corresponding wheats-wheatgrass addition line II-29-2i, 8 corresponding wheats-correspondence of wheatgrass addition line 5106,9 is small
Wheat-wheatgrass addition line II-5-1,10 corresponding wheats-wheatgrass addition line II-4-2,11 corresponding wheats-wheatgrass addition line 5038,12
Correspondence wheat-wheatgrass addition line 5043,13 corresponding wheats-wheatgrass addition line II-21-2,14 corresponding wheats-wheatgrass addition line II-
21-6,15 corresponding wheats-wheatgrass addition line II-9-3,16 corresponding wheats-wheatgrass addition line II-30-5,17 corresponding wheat-wheatgrasses
Addition line II-1-3,18 corresponding wheats-wheatgrass addition line II-3-1,19 corresponding wheats-wheatgrass addition line II-7-1,20 correspondences are small
Wheat-wheatgrass addition line II-8-1,21 corresponding wheats-wheatgrass 6P long arm end systems, 22 corresponding wheats-wheatgrass 6P galianconism end system.
As a result show：Using the special primer pair, in wheatgrass Z559, each wheat-wheatgrass addition line, wheat-wheatgrass
It can be expanded in 6P long arm ends system, wheat-wheatgrass 6P galianconism end system and obtain specific band, and on wheat Fukuho not
It can expand and obtain specific band, the target sequence for illustrating the special primer pair is the molecular labeling of P genome specifics.
Reclaiming each specific band and be sequenced, sequencing result shows, the size of each specific band is 1045 ±
5bp。
Embodiment 3, the specificity of special primer pair
1st, the genomic DNA of each sample to be tested is extracted respectively.
2nd, respectively using each genomic DNA as template, the special primer constituted using AcPR1F and AcPR1R is to entering performing PCR
Amplification, obtains pcr amplification product.
3rd, each pcr amplification product for obtaining step 2 carries out 1% agarose gel electrophoresis, as a result sees Fig. 5.
In Fig. 5, the M correspondence correspondences of marker, 1 corresponding wheatgrass Z1842,2 corresponding wheatgrass Z559,3 corresponding wheatgrass Z1750,4 are small
Wheat China spring, 5 corresponding wheat Fukuho, 6 corresponding einkorn wheat MO4, 7 corresponding durum wheat capital DR3, 8 correspondingly carry Mo Feiwei
Wheat TI1, 9 corresponding shape of tail goatweed Ae14,10 corresponding aegilops tauschii Y93,11 corresponding Aegilops comosa Y258,12 correspondingly intend this
Inferior that de- goatweed Ae49,13 corresponding Aegilops umbelluata Y39,14 corresponding rye RM2161,15 corresponding Cultivate berley Zhejiang skins 1,
The false roegneria kamoji Z1365,19 corresponding roegneria kamoji Z2192 of the correspondence of 16 corresponding haynaldia villosa Z1731,17 corresponding Leymus racemosus R429,18.
As a result show, AcCR1F and AcCR1R composition special primer to the A in Tribe Triticeae, B, D, S, C, G, M, U, R,
The genomes such as H, V, St, Ns, Y are without amplification, high specificity.
Embodiment 4, special primer pair and the performance comparision for compareing primer pair
1st, the genomic DNA of each sample to be tested is extracted respectively.
2nd, respectively using each genomic DNA as template, the special primer constituted using AcPR1F and AcPR1R is to entering performing PCR
Amplification, obtains pcr amplification product.
3rd, each pcr amplification product for obtaining step 2 carries out 1% agarose gel electrophoresis, as a result sees Fig. 6.
In Fig. 6, M correspondences marker, 1 corresponding wheatgrass Z559,2 corresponding wheats-wheatgrass 6P disomic addition lines 4844-12 are small
Wheat-wheatgrass addition line 5113,4 corresponding wheats-wheatgrass addition line II-9-3,5 corresponding wheats-wheatgrass addition line II-5-1,6 correspondences
Wheat-wheatgrass addition line 5043,7 corresponding wheat Fukuho, 8 corresponding timopheevi wheat TI1, 9 corresponding shape of tail goatweed Ae14,
10 corresponding rye RM2161,11 corresponding Ae.speltoides Ae49,12 corresponding haynaldia villosa Z1731.
As a result show, the special primer of AcPR1F and AcPR1R compositions is to equal on wheatgrass, each wheat-wheatgrass addition line
It can expand and obtain specific band, and can not be expanded in Tribe Triticeae each kind and obtain specific band, i.e., its target sequence is ice
Careless genome specific.
4th, respectively using each genomic DNA as template, entered using the ControlF and ControlR control primer pairs constituted
Performing PCR is expanded, and obtains pcr amplification product.
ControlF (sense primer)：5’-CCATTCTCTCGTGCAATGAC-3’；
ControlR (anti-sense primer)：5’-TCTCGGTTGAACCCAAGTTT-3’.
5th, each pcr amplification product for obtaining step 4 carries out 1% agarose gel electrophoresis, as a result sees Fig. 7.
In Fig. 7, M correspondences marker, 1 corresponding wheatgrass Z559,2 corresponding wheats-wheatgrass 6P disomic addition lines 4844-12 are small
Wheat-wheatgrass addition line 5113,4 corresponding wheats-wheatgrass addition line II-9-3,5 corresponding wheats-wheatgrass addition line II-5-1,6 correspondences
Wheat-wheatgrass addition line 5043,7 corresponding wheat Fukuho, 8 corresponding timopheevi wheat TI1, 9 corresponding shape of tail goatweed Ae14,
10 corresponding rye RM2161,11 corresponding Ae.speltoides Ae49,12 corresponding haynaldia villosa Z1731.
As a result show, the control primer pair of ControlF and ControlR compositions is added in wheatgrass, each wheat-wheatgrass
It can be expanded in system, Tribe Triticeae each kind and obtain band, specificity is very poor.
Claims (7)
1. a kind of special primer pair for being used to identify the inhereditary material whether vegetable material to be measured contains from wheatgrass, by sequence
Single strand dna shown in the sequence 2 of table and the single strand dna composition shown in the sequence 3 of sequence table.
2. special primer described in claim 1 is to identifying whether vegetable material to be measured contains in the inhereditary material of wheatgrass
Application.
3. a kind of detect the method whether vegetable material to be measured contains the inhereditary material from wheatgrass, comprise the following steps：With
The genomic DNA of vegetable material to be measured is template, using special primer described in claim 1 to entering performing PCR amplification, if obtained
Pcr amplification product, vegetable material to be measured contain the inhereditary material from wheatgrass, if not obtaining pcr amplification product, to be measured
Vegetable material does not contain the inhereditary material from wheatgrass.
4. a kind of DNA molecular, is following (a) or (b)：
(a) DNA molecular shown in the sequence 1 of sequence table from the nucleotides of 5 ' end 531-1575；
(b) DNA molecular shown in the sequence 1 of sequence table.
5. DNA molecular described in claim 4 is identifying whether vegetable material to be measured contains in the inhereditary material of wheatgrass
Using.
6. a kind of detect the method whether vegetable material to be measured contains the inhereditary material from wheatgrass, comprise the following steps：Adopt
With DNA molecular described in claim 4 as probe, treat measuring plants chromosome and carry out FISH, if with fluorescence
Signal, vegetable material to be measured contain the inhereditary material from wheatgrass, if be free of without fluorescence signal, vegetable material to be measured
There is the inhereditary material from wheatgrass.
7. DNA molecular shown in the sequence 1 of sequence table is used as the application of retrotransponsons.
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| CN102876801A (en) * | 2012-10-22 | 2013-01-16 | 中国科学院遗传与发育生物学研究所 | Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids |
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| CN1924017A (en) * | 2006-09-15 | 2007-03-07 | 中国农业科学院作物科学研究所 | Crested wheatgrass P genome specific sequence |
| CN102876801A (en) * | 2012-10-22 | 2013-01-16 | 中国科学院遗传与发育生物学研究所 | Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids |
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