CN107475390A - The exploitation and application of Thinopyrum ponticum tandem repetitive sequence specific probe - Google Patents
The exploitation and application of Thinopyrum ponticum tandem repetitive sequence specific probe Download PDFInfo
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Abstract
The invention discloses the exploitation and application of Thinopyrum ponticum tandem repetitive sequence specific probe.Common wheat, common wheat Thinopyrum ponticum translocation line and Thinopyrum ponticum are sequenced using specific length amplified fragments sequencing technologies (SLAF seq) by the present invention, analysis is compared by sequence data and obtains Thinopyrum ponticum distinguished sequence, and based on Thinopyrum ponticum distinguished sequence, develop tandem repetitive sequence specific probe.The tandem repetitive sequence specific probe of the present invention can be not only used for the Chang Miho couchgrass chromosome of Rapid identification decaploid and E. elongata and be identified to the translocation line during wheat transfer dyeing body fragment, and for molecular marker assisted selection improve wheat genetic characteristic important channel is provided, be also Wheat Molecular Breeding, phyletic evolution and germplasm identification important application foundation is provided.
Description
Technical field
The invention belongs to field of crop genetic breeding, and in particular to Thinopyrum ponticum tandem repetitive sequence is specifically visited
The exploitation and application of pin.
Background technology
Wheat (Triticum aestivm) is that cultivated area is maximum in the world, and yield is only second to the second generalized grain of corn
Crop.Nearly half a century, the total output of world wheat have increased more than twice, wherein excellent wheat breed is small to improving
Wheat yield has played conclusive effect.There is the advantageous genetic money that substantial amounts of wheat lacks in the nearly edge species of wheat wild
Source, fully develop and utilize these fine genes cultivation new variety of wheat and have become one of target of wheat breeding.It is long
Miho couchgrass (Thinopyrum ponticum, 2n=10x=70) is herbaceos perennial, belongs to grass family couchgrass
Category, is the important wild relatives of wheat.The merits such as there is long fringe to spend more for it, wide adaptability, resistance of reverse are strong, contain
Not available for many common wheats excellent genes (Shannon, 1978；Cox, 1991), and it is easily solid with wheat hybridizing, be
Improve the precious resources storehouse of existing wheat breed.In recent years, go deep into work, the Chang Miho intermedium disease-resistances of many new,
Adversity gene is constantly found so that it was recognized that what is developed at present is only wherein a little.Example
Such as, it contains wheat rust resistant gene, can resist the novel wheat stem rust germ Ug99 (Xu wreaked havoc in recent years in Africa
et al.,2009； Zheng et al.,2014)；It also evolves the tune that a set of efficiently control salinity absorbs, transports and secreted
Control mechanism, it is possible to increase wheat salt tolerance characteristic (Chen et al., 2004).
Transfer excellent genes are belonged to from wild relatives by distant hybridization and chromosome engineering and have proved to be wheat genetic
One fruitful approach of improvement.Early in the fifties China be just successfully made the hybridization of wheat and Genus Agropyron, wherein
Small wheat variety of laying down " No.6 Xiaoyan " annual planting area is once up to 670,000 hm2More than, and used in production and be up to 20 years.
In wheat-Chang Miho couchgrass filial generations, the alien chromatin that detection comes from E. elongata is asked as vital
Topic.Although some science of heredity means, such as Chromosomes Banding, in situ hybridization technology, can be used in Chang Miho Xia wheat genetic background
The detection of couchgrass inhereditary material, but these technologies are time-consuming, laborious, and the requirement to operation skill or instrument and equipment is also higher, and
And these laboratory facilities are unsuitable for carrying out high flux screening and scale identification.
Molecular marker assisted selection (Marker Assisted Selection) is to utilize and target gene close linkage
Molecular labeling or functional label, the genotype of Different Individual is differentiated exactly in generation after hybridization, and carried out accordingly auxiliary
Help the breeding technique of selection.Assisted Selection (MAS) is marked for profit using the special chemoattractant molecule mark of Chang Miho couchgrass chromosome
With couchgrass favorable genes, Wheat Molecular Breeding process is accelerated to be significant.Chang Miho couchgrass is derived from for example, utilizing
Anti- stem rust ospc gene Sr24, Sr25, Sr26 and Sr43 linked marker, the screening of Resistant gerplasm resource can be carried out, external source is lost
Identification, the positioning of disease-resistant gene of material are passed, is effectively improved the efficiency (Ayala- of molecular marker assisted selection breeding
Navarrete et al., 2007；Margo et al.,2005；Niu et al.,2014；Yu et al.,2010；Zheng
et al.,2014)。
Up to now, scientist develops E. elongata specific molecular marker using multiple technologies means, such as
Restrictive fragment length polymerphism (Restriction Fragment Length Polymorphism, RFLP) (Autrique
et al.,1995；Liu et al., 1999), AFLP (Amplified fragment length
Polymorphism, AFLP) (Prins et al., 2001；Zhang et al., 2008), DNArandom amplified polymorphic DNA
(Random Amplified Polymorphic DNA, RAPD) (Liu et al., 1998；You et al., 2002) it is, simple
Repetitive sequence (Simple sequence repeats, SSR) (Li et al., 2005；Jauhar et al.,2009；Shen
et al.,2004；You et al.,2002； You et al.,2003；Niu et al., 2014), sequence-tagged site
(Sequence Tagged Sites, STS) (Mago et al., 2005；Zhang et al., 2008), disease-resistant gene is similar
Substance markers (Resistance Gene Analogs Polymorphism, RGAP) (Chen et al., 2007), target site area
Amplification polymorphism (Target Region Amplified Polymorphism, TRAP) (Jauhar et al., 2009), suppression
Subtractive hybridization (Suppression Subtraction Hybridization, SSH) processed (Ge et al., 2012), digestion are expanded
Increasing polymorphic sequence (Cleaved Amplification Polymorphism Sequence, CAPS) (Shen et al.,
2004；Li et al., 2007), sequence specific amplification area (Sequence-characterized Amplified
Region, SCAR) (You et al., 2002；Prabhu et al., 2004；Yan et al.,2009).
However, the development in the Chang Miho couchgrass molecular labelings of , today of molecular biology rapid development relatively lags behind,
Mark source also focuses primarily upon its diploid kind.Because the Chang Miho couchgrass genome of decaploid is huge, in addition its genome with
Wheat volatiles have high homology, lack genom sequence information again so that the Chang Miho couchgrass molecular labeling of decaploid
Developmental research the shortcomings that time-consuming, cost is high, efficiency is low be present, the special chemoattractant molecule mark of chromosome can not be obtained in batches, seriously
Hinder the development and application of E. elongata genetic resources.
SLAF-seq (Specific-locus Amplified Fragment sequencing) is measured based on high pass
Extensive genotyping technique that sequence technology develops (Sun et al., 2013).The technology mainly passes through bioinformatics
Specific long fragment is screened in design, structure SLAF-seq libraries, and high-flux sequence obtains sequence information, software definition
With the step such as assess, screen the sequence label of magnanimity, special molecular labeling further developed according to sequence.Using SLAF-
Seq technologies can develop high density full-length genome molecular labeling, build high density genetic linkage mapses, carry out high-precision QTL and determine
Position.This method has the advantages that flux is high, accuracy is high, cost is low, the cycle is short, in Genes location, molecular breeding, germplasm money
The research fields such as source identification are applied.The applicable species scope of SLAF-seq technologies is wide, in crops, vegetables, forest, water
The Successful Practice in multiple species such as production (Zhang, et al., 2013).Using SLAF-seq technologies, 20 two are have successfully been obtained
The molecular labeling of times Chang Miho couchgrass 1E chromosomes of Ti and the Chang Miho couchgrass 7E chromosomes of 89 diploids (old scholar is strong etc.,
2013；Chen et al., 2013).It is effectively simplified to whole gene group, is uniformly distributed and is kept away in screening-gene group
The fragment for opening repetitive sequence is sequenced, and avoids the sequencing input to repetitive sequence region.When shortage species gene group information
When, it can equally utilize the technology to reduce species complexity, obtain reliable sequence label.Therefore, the hair of SLAF-seq technologies
The excavation of the special chemoattractant molecule mark of the exhibition Chang Miho couchgrass chromosomes of Dui brings new opportunity.
The content of the invention
First purpose of the present invention is to provide a kind of probe for being used to identifying or aiding in identification E. elongata chromosome.
Provided by the present invention for identifying or aiding in identify that the probe of E. elongata chromosome is following (1) or (2)：
(1) probe is the DNA molecular shown in sequence 135；
(2) probe is using the genomic DNA of E. elongata chromosome as template, using primer pair 67 or primer pair
63 or primer pair 65 expanded obtained DNA molecular；
The primer pair 67 is made up of the single strand dna shown in sequence 133 and the single strand dna shown in sequence 134；
The probe that the DNA molecular progress fluorescence labeling for being expanded to obtain using primer pair 67 obtains is named as into pThp5.121 to visit
Pin；
The primer pair 63 is made up of the single strand dna shown in sequence 125 and the single strand dna shown in sequence 126；
The probe that the DNA molecular progress fluorescence labeling for being expanded to obtain using primer pair 63 obtains is named as pThp3.81 probes；
The primer pair 65 is made up of the single strand dna shown in sequence 129 and the single strand dna shown in sequence 130；
The probe that the DNA molecular progress fluorescence labeling for being expanded to obtain using primer pair 65 obtains is named as pThp3.96 probes.
In above-mentioned probe, the probe is marked with fluorophor；The fluorophor is specially green fluorescence group.
Second object of the present invention is to provide the primer pair for identifying or aiding in identification E. elongata chromosome.
Provided by the present invention for identifying or aiding in identify that the primer pair of E. elongata chromosome is above-mentioned primer pair 67
Or primer pair 63 or primer pair 65.
In above-mentioned primer pair, the mole ratio of each bar primer in each primer pair is 1:1.
Third object of the present invention is to provide the PCR reagent for identifying or aiding in identification E. elongata chromosome.
PCR reagent provided by the invention includes above-mentioned primer pair.
In above-mentioned PCR reagent, final concentration of each bar primer in the PCR reagent in each primer pair is 10
μM。
Fourth object of the present invention is to provide the kit for identifying or aiding in identification E. elongata chromosome.
Include above-mentioned primer pair provided by the present invention for the kit for identifying or aiding in identify E. elongata chromosome
Or above-mentioned PCR reagent.
Mentioned reagent box also includes pAs1 probes；The pAs1 probes are the DNA molecular shown in sequence 136；The pAs1
Probe is marked with fluorophor；The fluorophor is specially red fluorescence group.
The 5th purpose of the present invention is to provide above-mentioned probe or above-mentioned primer pair or above-mentioned PCR reagent or mentioned reagent box
New application.
The invention provides above-mentioned probe or above-mentioned primer pair or above-mentioned PCR reagent or mentioned reagent box following 1) -12)
In it is any in application：
1) detect or aid in detection E. elongata chromosome or its fragment；
2) detection or auxiliary detection E. elongata chromosome or the product of its fragment are prepared；
3) detect or aid in detection to treat whether contain E. elongata chromosome or its fragment in measuring plants；
4) prepare detection or auxiliary detection treat in measuring plants whether the product containing E. elongata chromosome or its fragment；
5) identify or aid in identify that the E. elongata chromosome treated in measuring plants is the dyeing of which of 1-7 chromosomes
Body；
6) prepare and identify or aid in identify that the E. elongata chromosome treated in measuring plants is which of 1-7 chromosomes
The product of chromosome；
7) E. elongata chromosome or its fragment are followed the trail of；
8) tracking E. elongata chromosome or the product of its fragment are prepared；
9) molecular marker assisted selection breeding；
10) product of molecular marker assisted selection breeding is prepared；
11) Wheat Molecular Breeding；
12) product of Wheat Molecular Breeding is prepared.
In above-mentioned probe or above-mentioned primer pair or above-mentioned PCR reagent or mentioned reagent box or above-mentioned application, the long fringe is laid down
Wheat straw is Thinopyrum ponticum.
The 6th purpose of the present invention be to provide a kind of identification or auxiliary identification Thinopyrum ponticum chromosome or its
The method of fragment.
The method provided by the invention for identifying or aiding in identify Thinopyrum ponticum chromosome or its fragment is as follows
Method one or method two：
Method one：Identification or auxiliary identification treat whether contain Thinopyrum ponticum chromosome or its fragment in measuring plants
Method, with above-mentioned pThp5.121 probes or pThp3.81 probes or pThp3.96 probes with treating that it is in situ miscellaneous that measuring plants are carried out
Hand over, if there is hybridization signal, then it represents that treat to contain in measuring plants or candidate contains Thinopyrum ponticum chromosome or its piece
Section；
Method two：Identify or aid in identify that the Thinopyrum ponticum chromosome for treating to contain in measuring plants is No. 1-7 dye
The method of which of colour solid chromosome, with above-mentioned pThp5.121 probes or pThp3.81 probes or pThp3.96 probes and upper
State pAs1 probes and treat that measuring plants carry out in situ hybridization, according to the hybridization color of bands of a spectrum, position and deep or light to be measured come comprehensive descision
The Thinopyrum ponticum chromosome contained in plant is which of 1-7 chromosomes chromosome；
It is in situ miscellaneous that the in situ hybridization refers to that probe is carried out with plant root tip somatic mitosis division phases to be measured
Hand over；The in situ hybridization does not contain the step of blockading.
It is described to treat that measuring plants are the wheat platymiscium containing E. elongata chromosome or its fragment in the above method；Institute
Stating the wheat platymiscium containing E. elongata chromosome or its fragment, concretely octoploid of wheat-wheatgrass is " small to lay down 68 ".
" small lay down contains 12 (6 pairs) complete Chang Miho couchgrass chromosome of decaploid and 2 in 68 " to octoploid of wheat-wheatgrass
The Chang Miho couchgrass translocation chromosome of (1 pair) common wheat-decaploid.The repetitive sequence in close end spot distribution of the present invention
Probe pThp5.121 or pThp3.81 or pThp3.96 is in octoploid of wheat-wheatgrass " small complete Chang Miho in 68 " 8 (4 couples) that lays down
There is specific and incomplete phase on the Chang Miho couchgrass chromosome segments of in couchgrass chromosome and 2 (1 pair) translocation chromosomes
Same bands of a spectrum, and probe pAs1 is also distributed in the chromosome of wheat and its sibling species, is mainly distributed on D group chromosomes,
It is also distributed on the Chang Miho couchgrass chromosome of decaploid.Therefore, by the present invention probe pThp5.121 or pThp3.81 or
PThp3.96 and pAs1 probes are marked as the probe containing different fluorophors respectively, then by analyzing the color for hybridizing bands of a spectrum, position
Put with it is deep or light can comprehensive descision treat Thinopyrum ponticum chromosome in measuring plants for which of 1-7 chromosomes dye
Colour solid.Specific method is as follows：
If there are bright pThp5.121 hybridization signals in galianconism end；And galianconism and long-armed end and close end go out
Existing pAs1 hybridization signals, wherein, the pAs1 hybridization signals and pThp5.121 hybridization signal weights of galianconism end and close end appearance
The chromosome of conjunction is No. 1 chromosome (No. 1 chromosome is 1 pair of most long exogenous chromosome, is middle part centromere chromosome)；
If there are not pThp5.121 hybridization signals；And pAs1 occur miscellaneous for galianconism end, middle part and long-armed close end
Signal is handed over, and the pAs1 hybridization signals that galianconism occurs are significantly stronger than long-armed chromosome as No. 2 chromosomes (middle part centromere dye
Colour solid)；
If there are not pThp5.121 hybridization signals；And there are pAs1 hybridization signals in galianconism and long-armed close end, and
The chromosome that the pAs1 hybridization signals of long-armed appearance are significantly stronger than galianconism is No. 3 chromosomes (submedian centromere chromosome)；
If there are pThp5.121 hybridization signals in long-armed end；And there are obvious pAs1 hybridization signals in galianconism close end
Chromosome is No. 4 chromosomes (middle part centromere chromosome)；
If there are pThp5.121 hybridization signals in long-armed end；And there is pAs1 in galianconism and long-armed end and close end
Hybridization signal, and the pAs1 hybridization signal intensities occurred are close, wherein, the pAs1 hybridization signals that long-armed end occurs with
The chromosome that pThp5.121 hybridization signals overlap is No. 5 chromosomes (middle part centromere chromosome)；
If there is pThp5.121 hybridization signals, and the pThp5.121 hybridization letters that galianconism occurs in galianconism and long-armed end
Number slightly it is better than long-armed；And the chromosome for pAs1 hybridization signals do not occur is No. 6 chromosomes (submedian centromere chromosome)；
If pThp5.121 hybridization signals occurs in galianconism end, and there is the dyeing of 5 bright pAs1 hybridization signals in middle part
Body is No. 7 chromosomes (the variegated body of wheat-Chang Miho couchgrass transpositions).
Final object of the present invention is to provide a kind of decaploid for identifying or aiding in identification to treat to contain in measuring plants and grown
Fringe couchgrass chromosome is the product of which of 1-7 chromosomes chromosome.
It is provided by the invention to identify or aid in identify that the Thinopyrum ponticum chromosome for treating to contain in measuring plants is 1-7
The product of which of number chromosome chromosome includes above-mentioned probe pThp5.121 or pThp3.81 or pThp3.96 and above-mentioned
Probe pAs1.
The present invention utilizes specific length amplified fragments sequencing technologies (Specific-locus Amplified
Fragment sequencing, SLAF-seq) to common wheat, common wheat-Thinopyrum ponticum translocation line and ten times
Body E. elongata is sequenced, and comparing analysis by sequence data obtains Thinopyrum ponticum distinguished sequence, and with ten
Based on times body E. elongata distinguished sequence, the tandem repetitive sequence specific probe of the Chang Miho couchgrass of decaploid is developed.This
Invention tandem repetitive sequence specific probe can be not only used for the Chang Miho couchgrass chromosome of Rapid identification decaploid and long fringe is laid down wheat
Grass is identified to the translocation line during wheat transfer dyeing body fragment, and improves wheat genetic for molecular marker assisted selection
Characteristic offer important channel is also Wheat Molecular Breeding, phyletic evolution and germplasm identification provide important application foundation.
Brief description of the drawings
Fig. 1 is that the special chemoattractant molecule of Thinopyrum ponticum marks EA2-31 easy in common wheat-Thinopyrum ponticum
Position is the amplification in EA, China spring (CS) and Thinopyrum ponticum Th.ponticum (10x).M：MarkerII；
1：Common wheat-Thinopyrum ponticum translocation line EA；2：China spring (CS)；3：Thinopyrum ponticum
Th.ponticum (10x)。
Fig. 2 is octoploid of wheat-wheatgrass " small 68 " tip of a root body cell metaphase chromosome in situ hybridization figures of laying down.A is the long fringe of decaploid
Couchgrass whole genomic probe；B is that Thinopyrum ponticum specially changes fluorescence probe pThp5.121；C is pAs1 probes.
The pattern collection of illustrative plates that the hybridization bands of a spectrum that Fig. 3 is pThp5.121 and pAs1 are drawn.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Quantitative test in following embodiments, it is respectively provided with and repeats to test three times, results averaged.
Common wheat China spring (CS, 2n=6x=42) in following embodiments, Thinopyrum ponticum
" small 68 " (Xiaoyan 68,2n=8x=56) that lay down are in text for (Th.ponticum, 2n=10x=70) and octoploid of wheat-wheatgrass
Offer " Q.Zheng, Z.Lv, Z.Niu, B.Li, H.Li, S.S.Xu, F.Han, Z.Li, Molecular cytogenetic
characterization and stem rust resistance of five wheat-Thinopyrum ponticum
Mistake disclosed in partial amphiploids, J Genet Genomics 41 (11) (2014) 591-599. ", the public can be therefrom
The heredity of the academy of sciences of state is obtained with Developmental Biology research.
Common wheat-Thinopyrum ponticum translocation line (EA, 2n=6x=42) in following embodiments, pedigree are small
Laying down, 81/4/ small 81/3/ small 81//Shan 229/ of laying down of laying down is small to lay down 68, and it is " small to lay down 68 " that the translocation chromosome is present in octoploid of wheat-wheatgrass
In.Wherein, " small lay down 81 " is by Inst. of Genetics and Development Biology, CAS's seed selection, passes through Hebei province's agriculture within 2005
The crop varieties committee authorizes, authorization numbering：Wheat No. 2005006 is examined in Ji, and " Shan 229 " is made by Shaanxi Shanxi Academy of Agricultural Sciences grain
Institute's seed selection, authorized by the variety of crops committee of Jiangsu Province within 1994, authorization numbering：The careful word 227 of Soviet Union kind.
The exploitation and application of the special chemoattractant molecule mark of embodiment 1, Thinopyrum ponticum and fluorescence probe
First, the exploitation and application of the special chemoattractant molecule mark of Thinopyrum ponticum
1st, the extraction and purifying of genomic DNA
It is " small to lay down that the Chang Miho couchgrass of common wheat China spring, decaploid, octoploid of wheat-wheatgrass are extracted respectively using CTAB methods
68 " and the genomic DNA of the Chang Miho couchgrass translocation line of common wheat-decaploid, concrete operation step is as follows：
(1) appropriate tender tissue is taken, rapid grind into powder and is fitted into after being freezed in liquid nitrogen in 2mL centrifuge tubes.
(2) add 65 DEG C preheating the μ L of CTAB extract solutions 800, acutely vibration, 65 DEG C of water-bath 40-60min, or gently shake
It is even.
(3) centrifuge tube is taken out, often pipe adds 800 μ L chloroforms:Isoamyl alcohol (24:1), turn upside down abundant mixing, room temperature,
12000rpm/min, centrifuge 15min.
(4) the μ L of Aspirate supernatant about 600 are transferred to new 1.5mL centrifuge tubes, add isometric isopropanol, overturn and mix ,-
20 DEG C of low temperature place 30min.
(5) room temperature, 12000rpm/min centrifugation 10min, abandons supernatant.
(6) plus the μ L of 75% ethanol 700, washing of turning upside down precipitate, and 4 DEG C, 10000rpm centrifugation 5min, abandon supernatant.
(7) 65 DEG C of oven drying 3-5min, it is dissolved in appropriate 1 × TE containing RNase (10mg/mL), 37 DEG C of digestion RNA
1h, electrophoresis detection DNA quality, -20 DEG C of preservations.
2nd, distinguished sequence label is obtained based on SLAF-seq technologies
With SLAF-seq method to common wheat China spring, the Chang Miho couchgrass of decaploid, the Chang Miho of common wheat-decaploid
The genomic DNA of couchgrass translocation line is sequenced and (completed by Beijing Biomarker Technologies Co., Ltd.), obtains each sample
Sequence label.Using Blast methods by the Chang Miho couchgrass translocation line sequence label of common wheat-decaploid and China spring sequence
Column label and known Wheat volatiles sequence are compared, and reject and sequence mark of the wheat sequence similarity more than 50%
Label；Remaining sequence label is compared with the Chang Miho couchgrass sequence label of decaploid again, obtains similarity more than 50%
Sequence label is the special SLAF sequence labels of Thinopyrum ponticum.The special sequence of Thinopyrum ponticum is finally obtained altogether
Column label 585.
3rd, the exploitation of the special chemoattractant molecule mark of Thinopyrum ponticum
The sequence label obtained according to SLAF-seq technologies, any special SLAF sequence labels of selection 171 utilize
It is right that the softwares of Primer Premier 5.0 respectively design primer 1.In Sangon Biotech's synthetic primer,
Purified with HAP modes.
4th, PCR reacts
With common wheat-Thinopyrum ponticum translocation line (EA) and parent common wheat China spring (CS), decaploid
E. elongata (Th.ponticum, 2n=10x=70) is template, is set according to the E. elongata distinguished sequence that sequencing obtains
The primer of meter enters performing PCR amplification.PCR reaction systems are as shown in table 1, and PCR response procedures are as shown in table 2.
Table 1.PCR reaction systems
| PCR reactive components | Volume (μ L) |
| 2 × Taq Master Mix (Beijing CoWin Bioscience Co., Ltd.) | 10 |
| H2O | 6.6 |
| DMSO | 0.8 |
| Primers F (10 μm) | 0.8 |
| Primer R (10 μm) | 0.8 |
| DNA(150ng/μL) | 1 |
| Amount to | 20 |
Table 2.PCR response procedures
5th, the purifying and sequencing of agarose gel electrophoresis and PCR reaction products
Ago-Gel of the pcr amplification product 2%, electrophoresis is carried out under 120V burning voltages 20 minutes, electrophoretic buffer
For 1 × TAE.After electrophoresis terminates, observation result, photographic analysis are simultaneously purified (raw work bioengineering (Shanghai) to PCR primer
Limited company's kit) with being sequenced.If common wheat-E. elongata translocation line and Thinopyrum ponticum have expansion
Increase band, and China spring does not have amplified band, then it is assumed that this is labeled as the special chemoattractant molecule mark of Thinopyrum ponticum.
As a result show there are 67 pairs of primers in 171 pairs of primers in common wheat-E. elongata translocation line and the long fringe of decaploid
There is amplified band in couchgrass, and common wheat China spring does not have amplified band.67 long fringes of decaploid of the present invention are laid down
The special chemoattractant molecule mark of wheat straw is respectively provided with good accuracy, reliability and specialization, 67 Thinopyrum ponticum specialization point
Son mark is as shown in table 3.Fig. 1 be special chemoattractant molecule mark EA2-31 common wheat-Thinopyrum ponticum translocation line, in
Amplification in state's spring, Thinopyrum ponticum.As can be seen from Figure 1：EA2-31 is in common wheat-decaploid length
Expanded in fringe couchgrass translocation line (EA) and Thinopyrum ponticum and obtain the band that size is 500bp, and common wheat
China spring does not have amplified band.
The special chemoattractant molecule mark of 3. 67 Thinopyrum ponticums of table
The exploitation and application of embodiment 2, Thinopyrum ponticum tandem repetitive sequence specific probe
With Thinopyrum ponticum (Th.ponticum, 2n=10x=70) for template, using what is obtained in embodiment 1
67 special chemoattractant molecules mark to be expanded into performing PCR, obtains PCR primer.FISH probe is marked as after the PCR primer of gained is purified,
To octoploid of wheat-wheatgrass, " small 68 " tip of a root somatic mitosis division phases of laying down carry out in situ hybridization.Selection is grown in decaploid
There is specific hybridization signal on Miho couchgrass chromosomes, and the probe of amixia signal is as the Chang Miho of decaploid on chromosome of wheat
Couchgrass specially changes fluorescence probe.Simultaneously GISH analysis works are carried out to make probe using the Chang Miho couchgrass complete genome DNA of decaploid
To compare, the specific steps reference literature of probe progress GISH analyses is done using the Chang Miho couchgrass complete genome DNA of decaploid
“Q.Zheng, Z.Lv,Z.Niu,B.Li,H.Li,S.S.Xu,F.Han,Z.Li,Molecular cytogenetic
characterization and stem rust resistance of five wheat-Thinopyrum ponticum
Method in partial amphiploids, J Genet Genomics 41 (11) (2014) 591-599. ".
1st, the preparation of fluorescence probe
(1) Thinopyrum ponticum tandem repetitive sequence specific probe
Using Thinopyrum ponticum as template, the special chemoattractant molecule mark of 67 obtained in embodiment 1 is respectively adopted and carries out
PCR is expanded, and obtains PCR primer, and PCR primer after purification, is marked according to the system in table 4, and fluorescence spy is prepared respectively
Pin.Mark comprises the following steps that：
1) mark system is prepared according to the concentration in table 4 and component, wherein ,-the dNTPs of mark is Alexa Fluor-
488-dUTP (green glow, purchased from Invitrogen companies)；Unlabelled-dNTPs is purchased from Invitrogen companies；Archaeal dna polymerase
(concentration is 10U/ μ L) is purchased from Invitrogen companies；Dnase (concentration is 100mU/ μ L) is purchased from the full formula gold biotechnology in Beijing
Co., Ltd；Contain 500mM Tris, 50mM MgCl in 10 × Nick translation buffer (solvent is water)2, pH
=7.8.
2) blow and beat the mark system of step 1) acquisition repeatedly with pipette tips, mix, it is impossible to be vortexed, 15 DEG C are incubated 2 hours.It is placed in
Metal bath instrument (H203- PRO, China) in 15 DEG C react 2 hours.13000rpm centrifuges 30-40min, abandons supernatant, collects precipitation.
3) precipitation of a step 2) acquisition is washed with 70% ethanol and absolute ethyl alcohol respectively, lucifuge is dried.Then to precipitation
In plus 10 μ L buffer solutions (10 μ L buffer solutions are by 0.3M NaCl, 0.03M sodium citrates, 10mM Tris, 1mM EDTA and water group
Into chemical reagent is purchased from Chemical Reagent Co., Ltd., Sinopharm Group), make the final concentration of 200ng/ μ L of probe.
Table 4. marks system
(2) aegilops tauschii tandem repetitive sequence specific probe pAs1
Aegilops tauschii tandem repetitive sequence specific probe pAs1 is as shown in sequence 136, the same step of its preparation method (1), no
It is that pAs1 is marked using Texas-red-5-dCTP (feux rouges, purchased from PerkinElmer companies) with part.
2nd, in situ hybridization
(1) film-making of tip of a root somatic mitosis division phases
1)N2O handles root-tip cells
The seed of mature and plump is selected, is put into the culture dish for being lined with damp filter paper, is drenched with water and keeps certain wet
Degree, cultivates in 23 DEG C of constant incubators, when the tip of a root grows to 1-2 centimetres, the clip tip of a root, is put into the centrifuge tube of moistening, covers
It is put into after upper cover in air chamber and handles 2h, pressure is 10ATM (1.01Mpa).
2) tip of a root is fixed
90% acetic acid fixes the tip of a root 10 minutes, distilled water flushing 2 times, each 5min.
3) digest
The water on the tip of a root is somewhat blotted with filter paper, Root apical meristem is cut, is put into 20 μ L mixing enzyme solutions (1%
Pectase Y-23 and 2% cellulase Onozuka R-10 be dissolved in 1 × citrate buffer solution, be purchased from Japanese Japan
Yakult companies), 37 DEG C of water-bath 40-60min.
4) piece microscopy is dripped
The tip of a root 2 times after 70% alcohol rinse enzymolysis, remaining 200 μ L 70% alcohol in centrifuge tube, smash the tip of a root, low speed to pieces
10sec or so is centrifuged, alcohol is fallen to do, it is thin according to plant root tip size ethanol on the rocks (each tip of a root 20-40 μ L) suspension tip of a root
Born of the same parents；Clean slide is put into wet box, on 6-7 μ L root-tip cells hanging drop to slide, will cover lid, 5 minutes
Later microscopy, by chromosome sectioning Saving specimen in refrigerator it is standby.
(2) in situ hybridization
Slide to be hybridized is put into UV-crosslinked instrument (code-No.CL-1000, purchased from UVP companies of the U.S.), UV
Energy is 0.125J/cm2.Film-making is placed on ice, probe is prepared into hybridization solution, every slide with blockading by proper ratio
The upper plus μ L of hybridization solution 6, covered, 90 DEG C of denaturation 5min, hybridized overnight in 55 DEG C of wet box.Film-making is taken out, is put into 2 rapidly
Cover glass is slid in × SSC, the slice, thin piece back side is dried with blotting paper, anti-color fading agent is added dropwise and (contains DAPI, H-1200, purchased from U.S.
Vector Labs companies of state), after the cover glass for covering 24 × 50mm, detect, take a picture under fluorescence microscope.And according to statistics
Result, by exogenous chromosome according to sorting from big to small.
As a result common exploitation obtains following 3 E. elongatas and specially changes fluorescence probe：PThp5.121, pThp3.81 and
PThp3.96 probes.The SLAF sequences and primer of this 3 specialization fluorescence probes are shown in Table 5, and to specialization fluorescence probe in octoploid
" the small hybridization signal laid down on 68 " is analyzed (Fig. 2) for small wheat of laying down.As a result show：When utilization Thinopyrum ponticum gene
Group DNA makees probe, and common wheat China spring DNA is blockaded, and to octoploid of wheat-wheatgrass, " small 68 " root tip chromosomes of laying down carry out fluorescence
During in situ hybridization (Fig. 2A), probe and the ratio blockaded are 1:200,6 pairs of exogenous chromosomes and 1 from E. elongata
To having obvious hybridization signal containing wheat-E. elongata translocation chromosome.When the series connection weight specially changed using E. elongata
Complex sequences pThp5.121 do probe to octoploid of wheat-wheatgrass " it is small lay down 68 " carry out FISH when, totally 5 pairs dyeing body end
There is the hybridization signal of point-like in portion region.Wherein 1 pair of chromosome occurs the hybridization letter of point-like in long-armed and galianconism end
Number；There is the hybridization signal (Fig. 2 B) of point-like in long-armed or galianconism the end of 4 pairs of chromosomes.Again with from aegilops tauschii
Tandem repetitive sequence pAs1 makees probe and has carried out fluorescent hybridization (Fig. 2 C), by analyzing pThp5.121 probes and pAs1 probes
Hybridize the colors of bands of a spectrum, position and it is deep or light come comprehensive descision, further discriminate between octoploid of wheat-wheatgrass " small different length in 68 " of laying down
Fringe couchgrass chromosome.Repetitive probe pThp5.121 and pAs1 hybridization signal are clear and performance is stable, are dyed in external source
Distribution and abundance on body and chromosome segment is significantly different so that 6 pairs of exogenous chromosomes and 1 from E. elongata
To visibly different hybridization bands of a spectrum are presented containing wheat-E. elongata translocation chromosome, E. elongata can be clearly differentiated
In 1-7 chromosomes.The pattern collection of illustrative plates drawn according to probe pThp5.121 and pAs1 hybridization bands of a spectrum is as shown in figure 3, Fig. 3
Middle light gray chrominance signal is pAs1 hybridization sites, and dark-grey chrominance signal is pThp5.121 hybridization sites, black signal be pAs1 and
Hybridization site common pThp5.121.Octoploid of wheat-wheatgrass " external source dyeing of small 6 couple to lay down in 68 " from E. elongata
Body and 1 pair of specific testing result containing wheat-E. elongata translocation chromosome are as follows：
No. 1 chromosome is 1 pair of most long exogenous chromosome, is middle part centromere chromosome, is become clear in galianconism end
PThp5.121 hybridization signals；PAs1 hybridization signals positioned at long-armed and the end of galianconism and close end, wherein galianconism end and
The signal of close end overlaps with pThp5.121 hybridization signals.
No. 2 chromosomes are middle part centromere chromosome, no pThp5.121 hybridization signals；The short arm of a chromosome end, middle part with
And pAs1 hybridization signals occurs in long-armed close end, the signal of the short arm of a chromosome is significantly stronger than long-armed.
No. 3 chromosomes are submedian centromere chromosome, pThp5.121 hybridization signals do not occur；The short arm of a chromosome and
There are pAs1 hybridization signals in long-armed close end, and long-armed pAs1 hybridization signals will be significantly stronger than galianconism.
No. 4 chromosomes are middle part centromere chromosome, and pThp5.121 hybridization signals occurs in long-armed end；Galianconism close end
There are obvious pAs1 hybridization signals.
No. 5 chromosomes are middle part centromere chromosome, and pThp5.121 hybridization signals are located at long-armed end；PAs1 hybridization letters
Number positioned at long-armed and the end of galianconism and close end, signal intensity is close, wherein the pAs1 hybridization signals of long-armed end with
PThp5.121 hybridization signals overlap.
No. 6 chromosomes are submedian centromere chromosome, and pThp5.121 hybridization signals are respectively positioned at the long-armed and end of galianconism
Portion, the pThp5.121 hybridization signals of galianconism are slightly better than long-armed, pAs1 hybridization signals do not occur.
No. 7 chromosomes are the variegated bodies of wheat-Chang Miho couchgrass transpositions, and pThp5.121 hybridization signals are located at translocation chromosome
Galianconism end, i.e. E. elongata chromosome segment position；It is miscellaneous that 5 bright pAs1 occurs in the middle part of translocation chromosome
Signal is handed over, can determine whether that the fragment is common wheat 4DS4DL according to signal banding pattern.According to results of hybridization, the translocation chromosome
T can be named asTh-4DS·4DL.
There was no significant difference with pThp5.121 for specialization fluorescence probe pThp3.81 and pThp3.96 in situ hybridization result,
It is used equally for identifying or aids in identifying that measuring plants are treated in Thinopyrum ponticum chromosome or its fragment and identification or auxiliary identification
In Thinopyrum ponticum chromosome be which of 1-7 chromosomes chromosome.
The E. elongata of table 5. specially changes fluorescence probe SLAF sequences and primer
Sequence table
<110>Inst. of Genetics and Development Biology, CAS
<120>The exploitation and application of Thinopyrum ponticum tandem repetitive sequence specific probe
<160> 134
<210> 1
<211> 25bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
atctgtatcc ctagagtcgc tcatc 25
<210> 2
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
ccacttcttg gtgtagcagt gac 23
<210> 3
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
acacccgcca ctgctaagc 19
<210> 4
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
ccatcactcc cacaacccat c 21
<210> 5
<211> 24bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
aggaaccttg aatttatggc acag 24
<210> 6
<211> 24bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
tgacaagtcg tgcagatttt cact 24
<210> 7
<211> 24bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
cagattatgt ggacttactc ggca 24
<210> 8
<211> 24bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
tccaaaacct gttgtctaga gcct 24
<210> 9
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
cgaaagactg gcacggataa g 21
<210> 10
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
tgagtcggca ataccccaac 20
<210> 11
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
actaaaggaa catgcggtca cac 23
<210> 12
<211> 24bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
ttgttagtga cttaacggtg gcac 24
<210> 13
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
agtttcaggt tccgacagaa t 21
<210> 14
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 14
cagttgttct tgctgtggct 20
<210> 15
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 15
gttgataatc ttggagatga t 21
<210> 16
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 16
tgacaaactc atgcctctag g 21
<210> 17
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 17
ccattcatgt cggaccaaaa 20
<210> 18
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 18
cgctgcatgg cgtgattgat 20
<210> 19
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 19
gaagagaagg agaaggacgg g 21
<210> 20
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 20
tgtaatgact aggtgtttgg c 21
<210> 21
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 21
tcaggtctcg gcatctcatc t 21
<210> 22
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 22
ttatatttcc ctttgtttgc a 21
<210> 23
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 23
ggtttgagaa aaacaacatg a 21
<210> 24
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 24
atcgatcaca aggagaaacg 20
<210> 25
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 25
accgtggggg tggataag 18
<210> 26
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 26
cgagaaactg cattaagtaa a 21
<210> 27
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 27
ctcagtggta gcagcctc 18
<210> 28
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 28
cagcgacttg caagtcttgc 20
<210> 29
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 29
catcaccaca tggctttcaa 20
<210> 30
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 30
taatattttg actagcatgc g 21
<210> 31
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 31
aaagacaaag acagtgatta c 21
<210> 32
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 32
tcataatatc atgtatgcc 19
<210> 33
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 33
cagcccagag aattttttt 19
<210> 34
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 34
agtgaggttt acttaagcaa c 21
<210> 35
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 35
ttatagcgaa ggtcaccgta c 21
<210> 36
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 36
cgttcatgat tggatcagaa g 21
<210> 37
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 37
agctgctgca accccagcct 20
<210> 38
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 38
cacgtctaac agccaacagt t 21
<210> 39
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 39
gaagatacgc tatcggttca a 21
<210> 40
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 40
cgttcataaa caacggagca 20
<210> 41
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 41
tacatttgat cagctgagca acc 23
<210> 42
<211> 24bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 42
aaacataact gtggtttagt aaag 24
<210> 43
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 43
ctgatcttcg tcgtgggtca t 21
<210> 44
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 44
agtgtacatc cactcacaat cc 22
<210> 45
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 45
aggtgttctg tgcactactc tg 22
<210> 46
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 46
aagaaggtgg tatgccctct ta 22
<210> 47
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 47
caggttagtt ggtgatttat tt 22
<210> 48
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 48
atgtgcattt atgttcacag a 21
<210> 49
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 49
ggctcctctc ttttgagggg tta 23
<210> 50
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 50
agtattacaa agaaaacaaa ga 22
<210> 51
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 51
aagaaggtga cgcgtttttt tc 22
<210> 52
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 52
tagctgttaa gctacagcat ag 22
<210> 53
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 53
acacatgacc ctccaactgc a 21
<210> 54
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 54
ttgaagatgt gaatgccaat c 21
<210> 55
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 55
actgatgttt tactttttta ttt 23
<210> 56
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 56
atcactgtgt ctcaatgctt cca 23
<210> 57
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 57
gctagtgggt ggacctgctc 20
<210> 58
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 58
ccatgctcgc cagcacct 18
<210> 59
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 59
cttgctctag agcaaaaact ag 22
<210> 60
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 60
ttacacgtac ctttcacaaa gca 23
<210> 61
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 61
agggtgatat acagcctatc 20
<210> 62
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 62
cacccctaat taaacctgtt tag 23
<210> 63
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 63
tgcttcaatg cttgctcatc 20
<210> 64
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 64
ttaaggtttt ttttttgggg tag 23
<210> 65
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 65
acaagagttg gggtctgata tgg 23
<210> 66
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 66
aggttcaact aaggatccat c 21
<210> 67
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 67
agggggattt acagcctatc taa 23
<210> 68
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 68
cacccctaat taacccaa 18
<210> 69
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 69
cctataaatc aaatatcgtc tag 23
<210> 70
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 70
agtgttcccc aaatatccat ctc 23
<210> 71
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 71
acaataagac cactcgacaa a 21
<210> 72
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 72
gtattaatga ggtcaaaaag tgt 23
<210> 73
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 73
tggtttgcga cacttgaa 18
<210> 74
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 74
tgagttccag gctgcttttc at 22
<210> 75
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 75
tttttcattt ttcctttttc tgt 23
<210> 76
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 76
ctctgtaaca ccccatgtca tt 22
<210> 77
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 77
tcccattcgg cgaggcttca tta 23
<210> 78
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 78
ctttgggtga gatcatagaa tca 23
<210> 79
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 79
ctgttgactg gtcagctga 19
<210> 80
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 80
tcgggagcta cggcgacgt 19
<210> 81
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 81
attctctgga catgcattgt tgc 23
<210> 82
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 82
cacattgcaa atggatggga 20
<210> 83
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 83
atgtatatcg aaggcaaaca tac 23
<210> 84
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 84
actttgtagt tagaaacaga gga 23
<210> 85
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 85
ggcatcccat tcttcccgta 20
<210> 86
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 86
tctatatgta cctgcttcaa 20
<210> 87
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 87
ccatgagagc gaagatgat 19
<210> 88
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 88
acttgtgcat atttgtaaaa tgt 23
<210> 89
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 89
ttagattgta acgcaacctg gg 22
<210> 90
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 90
aacgacatta tcttgctttg ct 22
<210> 91
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 91
aaaggacgac tataagggtc aca 23
<210> 92
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 92
tgataggaat gttttggtga gg 22
<210> 93
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 93
aggttcacct caggatccat ct 22
<210> 94
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 94
aaaagatttg gggtctaatg cag 23
<210> 95
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 95
cctcttcttt ctcttagttg ccc 23
<210> 96
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 96
atgtattcat ctcttgggta c 21
<210> 97
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 97
tgataaaccc tccgctgcca a 21
<210> 98
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 98
agcctcgttg agcccagt 18
<210> 99
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 99
ctccgagttc caaacggcta 20
<210> 100
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 100
agtgtacgac tcctctgggc att 23
<210> 101
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 101
cttagttata tgcaaactta gca 23
<210> 102
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 102
ctctaatgcc atccatgtcg aa 22
<210> 103
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 103
ccaggggtct cttttccctc ata 23
<210> 104
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 104
acagtcatgc ttgatacaag cag 23
<210> 105
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 105
aaaagatttg gggtctaatg cag 23
<210> 106
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 106
aggttcacct caggatccat ct 22
<210> 107
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 107
cagtctgagt ttactgtttt aaa 23
<210> 108
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 108
aggtgactca tgatcaacca ac 22
<210> 109
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 109
ctctcatgcc atccatgttg aa 22
<210> 110
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 110
ccctccgcac tgcactctt 19
<210> 111
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 111
cactgtgaca cccggtttgt aat 23
<210> 112
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 112
aggggaggtt tcgtccacat t 21
<210> 113
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 113
gcctgtcaca catacttcag tcg 23
<210> 114
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 114
tgcagcttgt tcaggagagg 20
<210> 115
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 115
agtgaataaa ctaatagatc aaa 23
<210> 116
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 116
cacataactt cctcggattg c 21
<210> 117
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 117
atccatttcc cctacatcca ac 22
<210> 118
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 118
catttggggc gcattttt 18
<210> 119
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 119
tgcgtctcat ttgccacat 19
<210> 120
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 120
ggcgacgccg acggaagc 18
<210> 121
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 121
aaggaactag tccagatttc ca 22
<210> 122
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 122
accacaatga tcttgtgatt g 21
<210> 123
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 123
gttggcaccg tcaatggc 18
<210> 124
<211> 23bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 124
agactagtac tccctcggtt cca 23
<210> 125
<211> 18bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 125
cgtcggagag gcgtagcg 18
<210> 126
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 126
accgaactcc gtcacacgtt 20
<210> 127
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 127
atgtattcat ctcttgggta c 21
<210> 128
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 128
cctcttcttt ctcttagttg cc 22
<210> 129
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 129
atcctacgtc gcccggtaca aa 22
<210> 130
<211> 19bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 130
cccaaatccg ggatggtgt 19
<210> 131
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 131
gatataccgg agcattggga 20
<210> 132
<211> 21bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 132
acccacagac agactctgcc t 21
<210> 133
<211> 22bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 133
ttcacgggta actcatgttg gt 22
<210> 134
<211> 20bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 134
accggactcg gtgctacttg 20
<210> 135
<211> 381bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 135
ttcacgggta actcatgttg gtcgggtatc gatctccgtg ccgatccgac cgatgacgtg 60
ccgtgtgggg cgaaatttct ttttcccgcc caaaacctcg cctcccgcct caagtgccgt 120
ctcgcgccct cgcaccactg ttggatccct ccccttttga taaagtcctt tgcatcactg 180
ttggatccct cccctttttg tatttttgtt tagtgtcgcc ggggagacgc ggtgggagcg 240
gtgtgcaata gtacaccctg ttttggcatg gcggcggaat tttcccgccc gaaatggcga 300
aaaagtaacc acgccggcga gtgtctcaaa gcgctcccgg aggtgtaaaa acatgtatag 360
gcaagtagca ccgagtccgg t 381
<210> 136
<211> 1015bp
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 136
ggatccactt cgtgtacaaa acggacaatc tctttcaaag tatcaggatt tcatccggga 60
actcgtctgt tacaaaggga tttcattttt aaaacctatt tgaactcctg actttttgtg 120
tgttcaaaat gcaccattaa aagccacatc atcatttttc aatcctttct gacttcattt 180
gttatttttc atgcatttac taattatttt gagctataag accctaaaat tgaaaagcat 240
ttcaaatgaa ctctgaaaag gttgaaactt ggcatgatat catcaattca tccacatagc 300
atgtgcagga aagttgagag ggttatggca aaaactggat gcacttcgtg tacaaaacgg 360
acaatctctt tcaaagtatc aggatttcat ccggaaactc gtctgttaca aagggatttc 420
atttttttaa cttatttgaa ctcctgactt tttgtgtgtt caaaatgcac cattcaaagc 480
cacatcatca tttttcaatc ctttctgact tcatttgtta tttttcatgc atttactaat 540
tatttttagc tataagaccc taaaattgaa aagcatttca aatgaactct gaaaaggttg 600
aaacttggca tgatatcatc atttcatcca catagcatgt gcaagaaagt tgagagggtt 660
acggcaaaaa ctggatgcac ttcgtgtaca aaacggacaa tctctttcaa agtatcagga 720
tttcatccgg aaactcgtct gttacaaagg gatttcattt ttttaaactt acttgaactc 780
ctgacttttt gtgtgttcaa aatgcaccat tcaaagccac atcatcattt ttcaatcctt 840
tctgacttca tttgttattt ttcatgcatt tactaattat tttgagctat aagaccctaa 900
aattgaaaag catttccaat gaactctgaa aaggttgaaa gttggcatca tatcatcatt 960
tcatccacat agcatgtgca agaaatttga gagggttacg gcaaaaactg gatcc 1015
Claims (10)
1. the probe for identifying or aiding in identification E. elongata chromosome, for following (1) or (2)：
(1) probe is the DNA molecular shown in sequence 135；
(2) probe be using the genomic DNA of E. elongata chromosome as template, using primer pair 67 or primer pair 63 or
Primer pair 65 is expanded obtained DNA molecular；
The primer pair 67 is made up of the single strand dna shown in sequence 133 and the single strand dna shown in sequence 134；
The primer pair 63 is made up of the single strand dna shown in sequence 125 and the single strand dna shown in sequence 126；
The primer pair 65 is made up of the single strand dna shown in sequence 129 and the single strand dna shown in sequence 130.
2. probe according to claim 1, it is characterised in that：
The probe is marked with fluorophor；
Or, the fluorophor is specially green fluorescence group.
3. the primer pair for identifying or aiding in identification E. elongata chromosome, it is the primer pair 67 described in claim 1
Or primer pair 63 or primer pair 65.
4. the PCR reagent for identifying or aiding in identification E. elongata chromosome, including the primer pair described in claim 3.
5. the kit for identifying or aiding in identify E. elongata chromosome, including primer pair described in claim 3 or
PCR reagent described in claim 4.
6. kit according to claim 5, it is characterised in that：The kit also includes pAs1 probes；
The pAs1 probes are the DNA molecular shown in sequence 136；
Or, the pAs1 probes are marked with fluorophor；
Or, the fluorophor is specially red fluorescence group.
7. PCR reagent described in the primer pair or claim 4 described in probe or claim 3 described in claim 1 or 2 or
Kit described in claim 5 or 6 is following 1) -12) in it is any in application：
1) detect or aid in detection E. elongata chromosome or its fragment；
2) detection or auxiliary detection E. elongata chromosome or the product of its fragment are prepared；
3) detect or aid in detection to treat whether contain E. elongata chromosome or its fragment in measuring plants；
4) prepare detection or auxiliary detection treat in measuring plants whether the product containing E. elongata chromosome or its fragment；
5) identify or aid in identify that the E. elongata chromosome treated in measuring plants is which of 1-7 chromosomes chromosome；
6) prepare and identify or aid in identify that the E. elongata chromosome treated in measuring plants is the dyeing of which of 1-7 chromosomes
The product of body；
7) E. elongata chromosome or its fragment are followed the trail of；
8) tracking E. elongata chromosome or the product of its fragment are prepared；
9) molecular marker assisted selection breeding；
10) product of molecular marker assisted selection breeding is prepared；
11) Wheat Molecular Breeding；
12) product of Wheat Molecular Breeding is prepared.
8. PCR reagent described in the primer pair or claim 4 described in probe or claim 3 described in claim 1 or 2 or
The application described in kit or claim 7 described in claim 5 or 6, it is characterised in that：The E. elongata is ten times
Body E. elongata.
It is following method one or side 9. a kind of identify or aid in identification Thinopyrum ponticum chromosome or the method for its fragment
Method two：
Method one：Identification or auxiliary identification treat in measuring plants whether the side containing Thinopyrum ponticum chromosome or its fragment
Method, with the probe described in claim 1 or 2 and treat that measuring plants carry out in situ hybridization, if there is hybridization signal, then it represents that treat measuring plants
In contain or candidate contains Thinopyrum ponticum chromosome or its fragment；
Method two：Identify or aid in identify that the Thinopyrum ponticum chromosome for treating to contain in measuring plants is 1-7 chromosomes
Which of chromosome method, with the probe described in claim 1 or 2 and the pAs1 probes described in claim 6 with treating
Measuring plants carry out in situ hybridization, according to the hybridization color of bands of a spectrum, position and it is deep or light treat to contain in measuring plants come comprehensive descision ten
Times body E. elongata chromosome is which of 1-7 chromosomes chromosome；
The in situ hybridization does not contain the step of blockading.
10. a kind of identify or aid in identify that the Thinopyrum ponticum chromosome for treating to contain in measuring plants is 1-7 chromosomes
Which of chromosome product, it includes the pAs1 probes described in probe and claim 6 described in claim 1 or 2.
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| CN201710728863.4A CN107475390B (en) | 2017-08-23 | 2017-08-23 | Development and application of decaploid elytrigia elongata series repeat sequence specific probe |
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| CN110029188A (en) * | 2019-04-29 | 2019-07-19 | 四川农业大学 | A kind of Tetraploid Elytrigia 1E chromosome molecular labeling and its application |
| CN110106280A (en) * | 2019-05-30 | 2019-08-09 | 南京农业大学 | Oligonucleotide probe kit and usage thereof that is a kind of while detecting wheat and hundred Sa couchgrass chromosomes |
| CN114369679A (en) * | 2022-01-19 | 2022-04-19 | 中国科学院遗传与发育生物学研究所 | Development and application of decaploid elytrigia elongata molecular marker and tandem repeat probe thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396026A (en) * | 2018-04-28 | 2018-08-14 | 中国科学院遗传与发育生物学研究所 | The exploitation and application of the Chang Miho couchgrass indigo plant kernel Characters of decaploid special chemoattractant molecule label and fluorescence in situ hybridization probe |
| CN108396026B (en) * | 2018-04-28 | 2020-04-21 | 中国科学院遗传与发育生物学研究所 | Development and application of tenfold-length scion elytrigia repens blue-particle trait specialized molecular marker and fluorescent in-situ hybridization probe |
| CN110029188A (en) * | 2019-04-29 | 2019-07-19 | 四川农业大学 | A kind of Tetraploid Elytrigia 1E chromosome molecular labeling and its application |
| CN110106280A (en) * | 2019-05-30 | 2019-08-09 | 南京农业大学 | Oligonucleotide probe kit and usage thereof that is a kind of while detecting wheat and hundred Sa couchgrass chromosomes |
| CN110106280B (en) * | 2019-05-30 | 2022-06-10 | 南京农业大学 | Oligonucleotide probe kit for simultaneously detecting chromosomes of wheat and thinopyrum bessarabicum and using method thereof |
| CN114369679A (en) * | 2022-01-19 | 2022-04-19 | 中国科学院遗传与发育生物学研究所 | Development and application of decaploid elytrigia elongata molecular marker and tandem repeat probe thereof |
| CN114369679B (en) * | 2022-01-19 | 2024-04-02 | 中国科学院遗传与发育生物学研究所 | Ten-fold elytrigia elongata molecular marker and development and application of tandem repeat probe thereof |
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