CN105368821A - P genome acrocentric region specific Copia retrotransposon and applications thereof - Google Patents
P genome acrocentric region specific Copia retrotransposon and applications thereof Download PDFInfo
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Abstract
The present invention discloses a P genome acrocentric region specific Copia retrotransposon and applications thereof, wherein the P genome acrocentric region specific Copia retrotransposon is the following (a) or (b): (a) a DNA molecule represented by the nucleotides site 250-883 from the 5' terminal in the sequence 1, and (b) a DNA molecule represented by the sequence 1. The invention further discloses a specific primer pair comprising a single-stranded DNA molecule represented by the sequence 2 in the sequence list and the single-stranded DNA molecule represented by the sequence 3 in the sequence list. According to the present invention, the obtaining of the dispersed repetitive sequence pAcpCR1 provides the important effect for the research of the Agropyron cristatum plant evolution; a lot of wheat-Agropyron cristatum translocation lines are obtained, such that the new challenge is provided for the rapid detection of the exogenous Agropyron cristatum chromatin; and with the obtaining of the retrotransposon specific molecular marker based on the P genome acrocentric region, the efficient detection of the Agropyron cristatum acrocentric region P chromatin under the wheat background can be performed.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the special Copia retrotransponsons of the nearly centric region of a kind of P genome and application thereof.
Background technology
Repetitive dna sequence is the genomic chief component of most plants.It can be divided into two classes: tandem repetitive sequence and interspersed repetitive sequence.And most interspersed repetitive sequence has feature and the feature of transposon and retrotransponsons.This kind of dispersion reiterated DNA sequences not still Plant Genome important component part and also be Plant Genome evolve main power.Long end retrotransponsons is the focus of research in tumor-necrosis factor glycoproteins.In recent years, be separated and obtain some new interspersed repetitive sequence from plant, this is that better the understanding structure of retrotransponsons and the evolution of Plant Genome provide help.And in Agropyron, yet there are no the research of this respect.Be distributed in the interspersed repetitive sequence that the whole karyomit(e) of wheatgrass has a retrotransponsons feature so obtain one there is very important Evolutionary Significance.
As the genetic resources that wheat flour quality is important, Agropyron contains many high yield characteristics, as strong in tillering ability, spend more many small ears etc., has resistance to drought, the abiotic stress such as cold and the biotic such as Powdery Mildew, rust simultaneously.Li Lihui etc. utilize wheatgrass (A.cristatum) Z559 picking up from Xinjiang, China to hybridize with common wheat (TriticumaestivumL.) Fukuho, obtain multiple wheat-wheatgrass Alien disomic addition lines by backcrossing further with selfing.Genomic for wheatgrass P favorable genes is transferred to wheat and is provided prerequisite by being established as of the success of wheat-wheatgrass distant hybirdization and alien addition line.The acquisition of a large amount of translocation line is that effective utilization of wheatgrass favorable genes provides possibility, and the chromatinic tracking of wheatgrass and qualification just need simple and effective means.The acquisition of the interspersed repetitive sequence of genome specific sequence, especially genome specific and trace detection can be carried out as the molecular label of genome specific to the alien chromatin importing wheat based on PCR molecule marker of this exploitation.
In sum, just because of the meaning of retrotransponsons in Plant Evolution, and its vital role in wheat flour quality, be badly in need of in Agropyron, develop such interspersed repetitive sequence.
Summary of the invention
The object of this invention is to provide the special Copia retrotransponsons of the nearly centric region of a kind of P genome and application thereof.
Copia retrotransponsons (the molecule marker that the nearly centric region of P genome provided by the invention is special, called after specific DNA molecular), be following (a) or (b): the sequence 1 of (a) sequence table is from the DNA molecular shown in 5 ' end 250-883 position Nucleotide; The DNA molecular shown in sequence 1 of (b) sequence table.
The present invention also protects a species-specific primers pair, is made up of the single strand dna shown in the sequence 3 of the single strand dna shown in the sequence 2 of sequence table and sequence table.
The present invention also protects described special primer to identifying that whether vegetable material to be measured is containing the application derived from the genetic material of the nearly centric region of wheatgrass karyomit(e).
The present invention also protects and a kind ofly detects vegetable material to be measured whether containing the method for genetic material deriving from the nearly centric region of wheatgrass karyomit(e); comprise the steps: with the genomic dna of vegetable material to be measured as template; adopt described special primer to carrying out pcr amplification; if obtain pcr amplification product, vegetable material to be measured contains the genetic material deriving from the nearly centric region of wheatgrass karyomit(e), if do not obtain pcr amplification product, vegetable material to be measured may not containing the genetic material deriving from the nearly centric region of wheatgrass karyomit(e).
The size of described pcr amplification product can be 634 ± 5bp.
Whether the present invention also protects described specific DNA molecular at qualification vegetable material to be measured containing the application derived from the genetic material of the nearly centric region of wheatgrass karyomit(e).
The present invention also protects and a kind ofly detects vegetable material to be measured whether containing the method for genetic material deriving from the nearly centric region of wheatgrass karyomit(e); comprise the steps: to adopt described specific DNA molecular as probe; treat measuring plants karyomit(e) and carry out fluorescence in situ hybridization; if there is fluorescent signal, vegetable material to be measured contains the genetic material deriving from the nearly centric region of wheatgrass karyomit(e), if not there is fluorescent signal, vegetable material to be measured may not containing the genetic material deriving from the nearly centric region of wheatgrass karyomit(e).
Treat that measuring plants can be wheatgrass Z559, wheatgrass Z1842, wheatgrass Z1750, wheat China spring, wheat Fukuho, einkorn wheat MO described in arbitrary above
4, durum wheat capital DR
3, timopheevi wheat TI
1, shape of tail goatweed Ae14, aegilops tauschii Y93, Aegilops comosa Y258, Ae.speltoides Ae49, Aegilops umbelluata Y39, rye RM2161, E. elongata PI547326, cluster hair wheat Z1731, Leymus racemosus R429, false roegneria kamoji Z1365, roegneria kamoji Z2192, wheat-wheatgrass 6P disomic addition line 4844-12, wheat-wheatgrass 6P long arm end system, wheat-wheatgrass 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 addition line II-4-2, wheat-wheatgrass addition line 5038, wheat-wheatgrass addition line 5043, wheat-wheatgrass addition line II-21-2, wheat-wheatgrass addition line II-21-6, wheat-wheatgrass addition line II-9-3, wheat-wheatgrass addition line II-30-5, wheat-wheatgrass addition line 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 application of the DNA molecular shown in sequence 1 as retrotransponsons of sequence table.
Meaning of the present invention is as follows: the acquisition of (1) interspersed repetitive sequence pAcpCR1 has vital role for the evolution studying Wheatgrass; (2) due to the acquisition of a large amount of wheat-wheatgrass translocation line, make to propose new challenge to the chromatinic rapid detection of external source wheatgrass, and the P chromatin of nearly centric region can carry out efficient detection to the wheatgrass under Wheat Background based on the acquisition of the retrotransponsons specific molecular marker of the nearly centric region of P genome.
Accompanying drawing explanation
Fig. 1 is the FISH result of wheat-wheatgrass 6P disomic addition line 4844-12.
Fig. 2 is the FISH result of wheatgrass Z1842.
Fig. 3 is the electrophorogram of agarose gel electrophoresis in embodiment 2.
Fig. 4 is the electrophorogram of agarose gel electrophoresis in embodiment 3.
Fig. 5 be adopt AcpCR1F and AcpCR1R to form in embodiment 4 special primer to the electrophorogram carrying out pcr amplification agarose gel electrophoresis.
Fig. 6 is the electrophorogram that the contrast primer pair adopting ControlF and ControlR to form in embodiment 4 carries out pcr amplification agarose gel electrophoresis.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Wheatgrass Z559 (tetraploid; 4n=28): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; J.; X.Yang; H.Wang, H.Li, L.Li; X.Li, etal.2006.Theintrogressionofchromosome6Pspecifyingforinc reasednumbersoffloretsandkernelsfromAgropyroncristatumin towheat.TheoreticalandAppliedGenetics114:13-20.).
Wheatgrass Z1842 (diploid, 2n=14, P genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheatgrass Z1750 (hexaploid; 6n=42; P genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M., J.Zhang, J.Wang; X.Yang; A.Gao, X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat China spring (common wheat; hexaploid; 6n=42; ABD genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Yang Guohui; Yang Xinming, Wang Ruihui, Gao Ainong; Li Lihui, Liu Weihua .2010. wheat-wheatgrass addition line 14 is recombinated the restraining effect of P karyomit(e) to Ph gene. Science Bulletin: 463-467.).
Wheat Fukuho (common wheat; hexaploid; 6n=42; ABD genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Einkorn wheat MO
4(diploid; 2n=14; A genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M., J.Zhang, J.Wang; X.Yang; A.Gao, X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Durum wheat capital DR
3(tetraploid; 4n=28; AB genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M., J.Zhang, J.Wang; X.Yang; A.Gao, X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Timopheevi wheat TI
1(tetraploid; 4n=28; AG genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M., J.Zhang, J.Wang; X.Yang; A.Gao, X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Shape of tail goatweed Ae14 (diploid; 2n=14; C genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Aegilops tauschii Y93 (diploid; 2n=14; D genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Aegilops comosa Y258 (diploid; 2n=14; M genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Ae.speltoides Ae49 (diploid; 2n=14; S genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Aegilops umbelluata Y39 (diploid; 2n=14; U genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Rye RM2161 (diploid, 2n=14, R genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
E. elongata PI547326 (diploid; 2n=14; E genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Cluster hair wheat Z1731 (diploid; 2n=14; V genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Leymus racemosus R429 (diploid; 2n=14; Ns genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
False roegneria kamoji Z1365 (diploid; 2n=14; St genome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Roegneria kamoji Z2192 (diploid; 2n=14; Y gene group): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass 6P disomic addition line 4844-12 (there is 42 wheat bar karyomit(e)+1 pair of wheatgrass 6P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; J.; X.Yang; H.Wang; H.Li; L.Li; X.Li, etal.2006.Theintrogressionofchromosome6Pspecifyingforinc reasednumbersoffloretsandkernelsfromAgropyroncristatumin towheat.TheoreticalandAppliedGenetics114:13-20.).
Wheat-wheatgrass 6P long arm end system (there is 42 chromosomes of wheat+1 pair of wheatgrass 6P chromosome long arm): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass 6P galianconism end system (there is 42 chromosomes of wheat+1 pair of wheatgrass 6P the short arm of a chromosome): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass addition line 5113 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Han; H.; L.Bai; J.Su; J.Zhang; L.Song, A.Gao, etal.2014.GeneticrearrangementsofsixWheat – Agropyroncristatum6Padditionlinesrevealedbymolecularmark ers.PloSone9:e91066.).
Wheat-wheatgrass addition line 5114 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Han; H.; L.Bai; J.Su; J.Zhang; L.Song, A.Gao, etal.2014.GeneticrearrangementsofsixWheat – Agropyroncristatum6Padditionlinesrevealedbymolecularmark ers.PloSone9:e91066.).
Wheat-wheatgrass addition line II-26 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Han; H.; L.Bai; J.Su; J.Zhang; L.Song, A.Gao, etal.2014.GeneticrearrangementsofsixWheat – Agropyroncristatum6Padditionlinesrevealedbymolecularmark ers.PloSone9:e91066.).
Wheat-wheatgrass addition line II-29-2i (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Han; H.; L.Bai; J.Su; J.Zhang; L.Song, A.Gao, etal.2014.GeneticrearrangementsofsixWheat – Agropyroncristatum6Padditionlinesrevealedbymolecularmark ers.PloSone9:e91066.).
Wheat-wheatgrass addition line 5106 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Han; H.; L.Bai; J.Su; J.Zhang; L.Song, A.Gao, etal.2014.GeneticrearrangementsofsixWheat – Agropyroncristatum6Padditionlinesrevealedbymolecularmark ers.PloSone9:e91066.).
Wheat-wheatgrass addition line II-5-1 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass addition line II-4-2 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass addition line 5038 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass addition line 5043 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass addition line II-21-2 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass addition line II-21-6 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass addition line II-9-3 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre (Wu; M.; J.Zhang; J.Wang; X.Yang; A.Gao; X.Zhang, etal.2010.Cloningandcharacterizationofrepetitivesequence sanddevelopmentofSCARmarkersspecificforthePgenomeofAgrop yroncristatum.Euphytica172:363-372.).
Wheat-wheatgrass addition line II-30-5 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass addition line II-1-3 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass addition line II-3-1 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass addition line II-7-1 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
Wheat-wheatgrass addition line II-8-1 (there is 42 chromosomes of wheat+1 pair of wheatgrass P karyomit(e)): Institute of Crop Science, Chinese Academy of Agricultural Science's crop germplasm resource protection and research centre.
The discovery of embodiment 1, pAcpCR1 and the distribution characteristics on wheatgrass karyomit(e) thereof
One, the discovery of pAcpCR1
With wheat-wheatgrass 6P galianconism end system for material, obtained the DNA sequencing fragment of 6P galianconism by the method for micro-dissections and DOP-PCR.These sequences are connected to pMD19-T carrier, import in intestinal bacteria, obtain the DNA sequencing fragment storehouse that 6P is short.Wheatgrass Z559 is filtered out and the discrepant DNA clone of wheat Fukuho checks order with dot hybridization.
Analyze through BLAST, to there is a length be 1742bp, GC content be 43.8% DNA fragmentation, by this DNA fragmentation called after pAcpCR1, as shown in the sequence 1 of sequence table.
Long end (LTR) the Copia class retrotransposon of pAcpCR1 in 5 ' end 1041-1664 position Nucleotide and wheat 3B-specific B AC storehouse has at most the sequence similarity of 82%, and this wheat retrotransposon is a part of cgt0528b.
Two, the distribution characteristics of pAcpCR1 on wheatgrass karyomit(e) and specificity verification
Take pAcpCR1 as probe, respectively FISH (fluorescence in situ hybridization) is carried out to wheat-wheatgrass 6P disomic addition line 4844-12 and wheatgrass Z1842 and analyze.
The FISH of wheat-wheatgrass 6P disomic addition line 4844-12 the results are shown in Figure 1.Result shows, pAcpCR1 has distribution in both sides, 6P karyomit(e) nearly kinetochore, do not distribute in 6P karyomit(e) end, and on 42 karyomit(e)s in wheat source equal no signal.
The FISH of wheatgrass Z1842 the results are shown in Figure 2.PAcpCR1 all has distribution in both sides, 14 karyomit(e) nearly kinetochores, does not all distribute in each karyomit(e) end.
Result shows: pAcpCR1 is distributed in both sides, wheatgrass all chromosomal kinetochores, take pAcpCR1 as the chromatin that probe can identify the nearly centric region of wheatgrass.
The exploitation that embodiment 2, special primer are right
1, special primer is devised to as follows based on pAcpCR1:
AcpCR1F (sequence 2 of sequence table): 5 '-GCAAAGCTGACCAGATGACA-3 ';
AcpCR1R (sequence 3 of sequence table): 5 '-TCGAAAGAGCAAAAGCACAA-3 '.
2, the genomic dna of each sample to be tested is extracted respectively.
3, respectively with each genomic dna for template, adopting the special primer that obtains of step 1 to carrying out pcr amplification, obtaining pcr amplification product.
4, each pcr amplification product step 3 obtained carries out 1% agarose gel electrophoresis, the results are shown in Figure 3.
In Fig. 3, the corresponding marker of M, 1 corresponding wheatgrass Z559, 2 corresponding wheat Fukuho, 3 corresponding wheats-wheatgrass 6P disomic addition line 4844-12, 4 corresponding wheat-wheatgrass addition lines 5113, 5 corresponding wheat-wheatgrass addition lines 5114, 6 corresponding wheats-wheatgrass addition line II-26, 7 corresponding wheats-wheatgrass addition line II-29-2i, 8 corresponding wheat-wheatgrass addition lines 5106, 9 corresponding wheats-wheatgrass addition line II-5-1, 10 corresponding wheats-wheatgrass addition line II-4-2, 11 corresponding wheat-wheatgrass addition lines 5038, 12 corresponding wheat-wheatgrass addition lines 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 wheats-wheatgrass addition line II-1-3, 18 corresponding wheats-wheatgrass addition line II-3-1, 19 corresponding wheats-wheatgrass addition line II-7-1, 20 corresponding wheats-wheatgrass addition line II-8-1, 21 corresponding wheats-wheatgrass 6P long arm end system, 22 corresponding wheats-wheatgrass 6P galianconism end system.
Result shows: adopt described special primer pair, wheatgrass Z559, each wheat-wheatgrass addition line, wheat-wheatgrass 6P long arm end system, wheat-wheatgrass 6P galianconism end system all can increase and obtain specific band, and can not increase on wheat Fukuho and obtain specific band, described special primer is described right target sequence is the molecule marker of P genome specific.
Reclaim each specific band and check order, sequencing result shows, the size of each specific band is 634 ± 5bp.
The specificity that embodiment 3, special primer are right
1, the genomic dna of each sample to be tested is extracted respectively.
2, respectively with each genomic dna for template, adopt AcpCR1F and AcpCR1R composition special primer to carrying out pcr amplification, obtain pcr amplification product.
3, each pcr amplification product step 2 obtained carries out 1% agarose gel electrophoresis, the results are shown in Figure 4.
In Fig. 4, M corresponding marker, 1 corresponding wheatgrass Z1842,2 corresponding wheatgrass Z559,3 corresponding wheatgrass Z1750,4 corresponding wheat China spring, 5 corresponding wheat Fukuho, 6 corresponding einkorn wheat MO
4, 7 corresponding durum wheat capital DR
3, 8 corresponding timopheevi wheat TI
19 corresponding shape of tail goatweed Ae14,10 corresponding aegilops tauschii Y93,11 corresponding Aegilops comosa Y258,12 corresponding Ae.speltoides Ae49,13 corresponding Aegilops umbelluata Y39,14 corresponding rye RM2161,15 corresponding E. elongata PI547326,16 corresponding cluster hair wheat Z1731,17 corresponding Leymus racemosus R429,18 corresponding false roegneria kamoji Z1365,19 corresponding roegneria kamoji Z2192.
Result shows, AcpCR1F and AcpCR1R composition special primer to genomes such as A, B, D, S, C, G, M, U, R, E, V, St, Ns, the Y in Tribe Triticeae without amplification, high specificity.
Embodiment 4, special primer pair and the Performance comparision contrasting primer pair
1, the genomic dna of each sample to be tested is extracted respectively.
2, respectively with each genomic dna for template, adopt AcpCR1F and AcpCR1R composition special primer to carrying out pcr amplification, obtain pcr amplification product.
3, each pcr amplification product step 2 obtained carries out 1% agarose gel electrophoresis, the results are shown in Figure 5.
In Fig. 5, the corresponding marker of M, 1 corresponding wheatgrass Z559,2 corresponding wheats-wheatgrass 6P disomic addition line 4844-12, the corresponding wheat of wheat-wheatgrass addition line 5113,4-wheatgrass addition line II-9-3,5 corresponding wheats-wheatgrass addition line II-5-1, the corresponding wheat Fukuho of 6 corresponding wheat-wheatgrass addition line 5043,7,8 corresponding timopheevi wheat TI
1, 9 corresponding shape of tail goatweed Ae14,10 corresponding rye RM2161,11 corresponding Ae.speltoides Ae49,12 corresponding cluster hair wheat Z1731.
Result shows, the special primer of AcpCR1F and AcpCR1R composition obtains specific band to all increasing on wheatgrass, each wheat-wheatgrass addition line, and can not increase in each kind of Tribe Triticeae and obtain specific band, namely its target sequence is wheatgrass genome specific.
4, respectively with each genomic dna for template, adopt ControlF and ControlR composition contrast primer pair carry out pcr amplification, obtain pcr amplification product.
ControlF (upstream primer): 5 '-TACTTGTTGCCAAAGGGGGA-3 ';
ControlR (downstream primer): 5 '-TTTGCCAATGCATGTCCTTA-3 '.
5, each pcr amplification product step 4 obtained carries out 1% agarose gel electrophoresis, the results are shown in Figure 6.
In Fig. 6, the corresponding marker of M, 1 corresponding wheatgrass Z559,2 corresponding wheats-wheatgrass 6P disomic addition line 4844-12, the corresponding wheat of wheat-wheatgrass addition line 5113,4-wheatgrass addition line II-9-3,5 corresponding wheats-wheatgrass addition line II-5-1, the corresponding wheat Fukuho of 6 corresponding wheat-wheatgrass addition line 5043,7,8 corresponding timopheevi wheat TI
1, 9 corresponding shape of tail goatweed Ae14,10 corresponding rye RM2161,11 corresponding Ae.speltoides Ae49,12 corresponding cluster hair wheat Z1731.
Result shows, the contrast primer pair of ControlF and ControlR composition all can increase and obtain band in wheatgrass, each wheat-wheatgrass addition line, each kind of Tribe Triticeae, and specificity is very poor.
Claims (7)
1. a species-specific primers pair, is made up of the single strand dna shown in the sequence 3 of the single strand dna shown in the sequence 2 of sequence table and sequence table.
2. special primer described in claim 1 is to identifying that whether vegetable material to be measured is containing the application derived from the genetic material of the nearly centric region of wheatgrass karyomit(e).
3. one kind is detected vegetable material to be measured whether containing the method for genetic material deriving from the nearly centric region of wheatgrass karyomit(e), comprise the steps: with the genomic dna of vegetable material to be measured as template, adopt special primer described in claim 1 to carrying out pcr amplification, if obtain pcr amplification product, vegetable material to be measured contains the genetic material deriving from the nearly centric region of wheatgrass karyomit(e), if do not obtain pcr amplification product, vegetable material to be measured may not containing the genetic material deriving from the nearly centric region of wheatgrass karyomit(e).
4. a DNA molecular, is following (a) or (b):
A the sequence 1 of () sequence table is from the DNA molecular shown in 5 ' end 250-883 position Nucleotide;
The DNA molecular shown in sequence 1 of (b) sequence table.
5. DNA molecular described in claim 4 is identifying that whether vegetable material to be measured is containing the application derived from the genetic material of the nearly centric region of wheatgrass karyomit(e).
6. one kind is detected vegetable material to be measured whether containing the method for genetic material deriving from the nearly centric region of wheatgrass karyomit(e), comprise the steps: to adopt DNA molecular described in claim 4 as probe, treat measuring plants karyomit(e) and carry out fluorescence in situ hybridization, if there is fluorescent signal, vegetable material to be measured contains the genetic material deriving from the nearly centric region of wheatgrass karyomit(e), if not there is fluorescent signal, vegetable material to be measured may not containing the genetic material deriving from the nearly centric region of wheatgrass karyomit(e).
7. the DNA molecular shown in sequence 1 of sequence table is as the application of retrotransponsons.
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| CN110055317A (en) * | 2019-04-29 | 2019-07-26 | 中国农业科学院作物科学研究所 | The method of external source function candidate gene is excavated in wheat distance edge hybrid offspring's Small piece transposition system |
| CN110055317B (en) * | 2019-04-29 | 2022-04-26 | 中国农业科学院作物科学研究所 | Method for discovering exogenous functional candidate genes in wheat distant hybridization progeny small-fragment translocation line |
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