CN109913578A - A kind of molecular labeling and its application of detection ovum fringe goatweed and wheat hybridizing HMW-GS gene - Google Patents

Abstract

The invention discloses molecular labeling and its applications of a kind of detection ovum fringe goatweed and wheat hybridizing HMW-GS gene, the molecular labeling is obtained by primer pair Ao-1Mx through PCR amplification, and the nucleotide sequence of the primer pair Ao-1Mx is as shown in NO.1~2 SEQ ID.Carrying out PCR amplification using the primer can rapidly and accurately detect in wheat distance edge hybrid offspring whether contain ovum fringe goatweed 1Mx type HMW-GS gene loci.This method can accurately detect that ovum fringe goatweed whether there is with 1Mx type HMW-GS gene in wheat hybridizing offspring, facilitate and tracks the gene, breeding work efficiency is greatly improved, the process of Wheat Breeding for Quality is accelerated, is had a good application prospect in Wheat Breeding for Quality field.

Description

It is a kind of detection ovum fringe goatweed and wheat hybridizing HMW-GS gene molecular labeling and its Using

Technical field

The present invention relates to molecular genetic technique field, it is related to a kind of detection ovum fringe goatweed and wheat hybridizing HMW-GS base The molecular labeling of cause and its application, specially ovum fringe goatweed and ovum fringe goatweed 1Mx type high score in wheat distance edge hybrid offspring The molecular labeling of son amount gluten subunit (high molecular weight glutenin subunit, HMW-GS) gene And its application.

Background technique

It, can be the excellent genes of the nearly edge wildlife species of wheat wild by distant hybridization in the genetic improvement process of wheat Common wheat is imported, and then changes the genetic constitution of wheat, therefore the correlated traits for effectively improveing wheat is widely used in In wheat breeding.Wild Related species of the Aegilops species as wheat, because having stripe rust resisting, leaf rust resistance, anti-stem rust Disease, mildew-resistance, the excellent character such as drought-enduring, saline-alkali tolerant, cold-resistant, degeneration-resistant and high protein is the important external source of genetic improvement of wheat Favorable genes library.High-molecular-weight glutelin subunit (HMW-GS) is the important storage protein of Wheat Endosperm, accounts for wheat and always stores 10% or so of albumen is the important determinant of wheat flour processing quality.In common wheat and its nearly edge species, high score Son amount gluten subunit is controlled by the individual gene site for being located at the homologous group's chromosome long arm of part 1, each HMW-GS It all include the gene of 2 close linkages on gene loci, middle-molecular-weihydroxyethyl is biggish to be known as X-type subunit, the lesser title of molecular weight For Y type subunit.Existing research shows that the type for the HMW-GS that common wheat itself contains is less, and in wheat relative genus object Contain a large amount of new HMW-GS variation types in kind.

Ovum fringe goat (Aegilops ovata L., U^oU^oM^oM^o, 2n=4x=28) and it is one of nearly edge species of wheat, tool There are many genes such as excellent mildew-resistance, stripe rust resisting, degeneration-resistant and quality, and are used as parent and pass through distant hybridization Mode carries out genetic improvement to common wheat.The new high-molecular-weight glutelin 1Mx type subunit base identified from ovum fringe goat Cause is very unique allelic variation type.Forefathers substitute strain by the chromosome specific of ovum fringe goatweed, it was demonstrated that 1Mx subunit has positive influence to wheat dough intensity.

During external source target gene imports wheat, the gene or chromosome segment being transferred are carried out accurate, fast The identification and tracking of speed, can be improved the validity of selection, so as to shorten breeding cycle, improve breeding efficiency.Therefore, to importing It will be distant hybridization breeding process that exogenous chromosome, chromosome segment or the entrained foreign gene of wheat, which carry out effectively identification, In highly important link.Currently, the common method of identification exogenous chromosome mainly includes Morphological Identification and cytological Identification. Morphological analysis compares hybrid plant and parent by observation and makes identification in the morphologic similarities and differences；Cytological Identification passes through Karyotyping, chromosome pairing analysis, C- band technology etc. identify hybrid.In addition, hybridization in situ technique is also used extensively In the identification of exogenous chromosome or segment.

Although being influenced by subjective factors such as experiences however, Morphological Identification is intuitive and certain objective traits can not lead to Morphologic appearance is crossed to come out and there is sizable limitation；And cytological Identification and in situ hybridization all must be by crossbreeding into Plant, it is stringent to season and materials time requirement, and it is more complex to analyze and identify method, to the requirement of technology, operation and practical experience Height, and because being the identification carried out from Chromosome level, due to the friendship in fission process there is chromosome segment Change and lose, be difficult to conclude objective trait gene whether necessary being and normal expression.Therefore, new in continuously implementation selection cross Kind (be) during material it is necessary to establish it is a kind of it is simple, efficiently, quickly detection ovum fringe goatweed and the remote edge of wheat are miscellaneous The method for handing over offspring 1Mx high-molecular wheat glutelin subunit gene.Accordingly, we are according to known ovum fringe goatweed high molecular weight 1Mx type glutelin sub-gene sequence, develops specific primer, not only identifies the hybridization with ovum fringe goatweed for parent Belong to the encoding gene band of ovum fringe goatweed 1Mx type subunit in progeny material, and this labeled primer is with very strong special Property (failing to expand purpose band in the wheat that uses in more than 40 parts of productions), can be from ovum using primer provided by the invention It is accurately tracked in fringe goatweed and common wheat filial generation and detects the transfer feelings containing 1Mx type glutelin sub-gene Condition, this will greatly facilitate the research work to distant hybrid progeny and accelerate the process that kind (is) breeding.

Summary of the invention

The purpose of the present invention is to provide 1Mx type high molecular weight paddy eggs in ovum fringe goatweed and wheat distance edge hybrid offspring The molecular labeling and its detection primer of Bai Yaji (HMW-GS) gene, another object are miscellaneous using the remote edge of primer progress wheat Hand over the identification of ovum fringe goatweed 1Mx type HMW-GS and the breeding of kind (being) in offspring.

In order to achieve the above technical purposes, the present invention is realized especially by following technical scheme:

The present invention utilizes ovum fringe goatweed 1Mx high molecular weight subunit gene order (GenBank accession number in ncbi database For KX375404.1) and other types high molecular weight subunit sequence include: 1Dx2 (Genbank accession number: KF466259.1), 1Dy3 (Genbank accession number: KF466260.1), 1Ax (Genbank accession number: JQ007589.1), 1Bx13 (Genbank Accession number: EF540764.1), 1Dy11 (Genbank accession number: EU528008.1), 1By16 (Genbank accession number: EF540765.1), 1Ay (Genbank accession number: MF098417.1), 1Uy (Genbank accession number: KX375407.1), 1Ux (Genbank accession number: KX375406.1) gene order, carries out Multiple Sequence Alignment analysis with DNAMAN7, identifies ovum fringe goat Special SNP site in careless 1Mx type HMW-GS gene order.Ovum fringe goatweed can be detected by devising a pair using these sites The specific primer of 1Mx type HMW-GS gene.Therefore in ovum fringe goatweed provided by the invention and wheat distance edge hybrid offspring The PCR molecular labeling expanding fragment length of 1Mx type HMW-GS gene is 182bp.

A kind of molecular labeling detecting ovum fringe goatweed and wheat hybridizing HMW-GS gene, the molecular labeling is by primer Ao-1Mx is obtained through PCR amplification, wherein primer pair Ao-1Mx is as follows:

Upstream primer sequence: 5 '-CGCCCTCGTGGCTCTCACCC-3 '；

Downstream primer sequence: 5 ' TTTGCTGCTGGTATTGTCCA-3 '.

Application of the molecular labeling in wheat hybridizing progeny selection is also within protection scope of the present invention.This point Son label can be used for screening the wheat hybridizing offspring containing ovum fringe goatweed 1Mx type HMW-GS gene.

In another aspect of this invention, the molecular labeling is in detection ovum fringe goatweed and wheat distance edge hybrid offspring Application in 1Mx type HMW-GS gene, specifically: PCR amplification material genomic DNA to be detected is carried out with primer Ao-1Mx, such as Fruit can amplify the amplified fragments of 182bp with primer Ao-1Mx, then explanation should be to ovoscopy fringe goatweed and wheat distance edge hybrid There are 1Mx type HMW-GS genes in offspring.

In another aspect of this invention, the present invention provides one kind for detecting ovum fringe goat in wheat distance edge hybrid offspring The primer of careless 1Mx type HMW-GS gene, the primer are Ao-1Mx.

Similarly, primer ovum fringe goatweed 1Mx type HMW-GS gene in detection wheat distance edge hybrid hybrid is answered With also within the scope of the present invention.

In another aspect of this invention, ovum fringe goatweed 1Mx type in a kind of detection wheat distance edge hybrid hybrid is provided The method of HMW-GS gene, comprising the following steps: carry out PCR method with primer Ao-1Mx and expand hybrid wheat genome to be checked DNA, if can amplify the amplified fragments of 182bp with primer Ao-1Mx, illustrating the wheat to be checked, there are ovum fringe goatweeds 1Mx type glutelin sub-gene.

Preferred pcr amplification reaction program are as follows: 94 DEG C of initial denaturation, 5min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C Extend 40s, totally 30 circulations；Extend 72 DEG C of 10 min eventually.

In another aspect of this invention, a kind of kit is provided, the kit contains primer Ao-1Mx, the reagent Box can be used for detecting ovum fringe goatweed 1Mx type HMW-GS gene in wheat distance edge hybrid offspring.

Preferably, the kit also contains positive control template, and the positive control template is to contain ovum fringe goat The plasmid of careless 1Mx type HMW-GS encoding gene.

The invention has the benefit that

The specific primer for the ovum fringe goatweed high molecular weight 1Mx type glutelin sub-gene that the present invention is developed, directly The augmentation detection that target gene is carried out to filial generation material, can not only do the true and false of filial generation from gene molecule level Judge out, and is capable of the transfer and presence of trace detection gene during continuously giving back cross breeding certainly, and identify Method is simple, as a result reliably, compensates for the extensive of Morphological Identification, cytological Identification it is cumbersome the deficiencies of, this is to passing through remote edge Crossbreeding will be a kind of good assistant breeding means to improve for common wheat.It, will using identification method of the invention Follow-up study is set to have more purpose and specific aim.

Detailed description of the invention

Fig. 1 is the specific primer of ovum fringe goatweed 1Mx type HMW-GS gene；

Fig. 2 is offspring (AS6/2/YY2//CM41F7) the SDS-PAGE electrophoresis detection figure being continuously selfed；

Fig. 3 is the detection figure that gene-specific amplification is carried out using Ao-1Mx primer pair parent and filial generation；Wherein, M For pUC19DNA/MspI (Hpall) Marker (stripe size is successively from lower to upper are as follows: 110,147,190,242,331,404, 501bp), AS6 is ovum fringe goatweed (hybridization maternal), and YY2 is heterologous No. 2, and CM41 is river wheat 41, AS6/2/YY2// CM41F7-1 to 18 is to obtain Progeny plants number in 7 generations of selfing；

Fig. 4 is the detection figure that gene-specific amplification is carried out using 46 wheat breeds (being) of Ao-1Mx primer pair；Wherein, M is pUC19DNA/MspI (Hpall) Marker, number 1-46 is respectively as follows: China spring, 27, river agriculture 32 is educated in good wheat No. 2, river, in 31, river wheat 602, western section wheat 518, river wheat 36, river 12147, Mianyang wheat 318, river are educated in section wheat 169, Chongqing 98767, river wheat 30, river Educate 17, Chuanmai 107,18, another name for Sichuan Province wheat 921, river agriculture 17, river wheat are educated in Nan Nong 02Y393, interior 2889, inland river 24066, another name for Sichuan Province wheat 1602, river 605, Zhou Mai 22, another name for Sichuan Province wheat 133, river wheat 58, river spoke 14, river wheat 42, continuous wheat 285, continuous wheat 602, river wheat 604, river wheat 64, river Wheat 104, river wheat 601, river wheat 1546, Hong Mai 161, Mianyang 335, another name for Sichuan Province wheat 969, river wheat 47, silk floss 06-458, river wheat 32, river agriculture 16, Educate 16, continuous wheat 51, river 07005, river wheat 1580 in river.

Specific embodiment

Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to In the scope of protection of the invention.

Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.

Material used in the present invention includes: ovum fringe goatweed AS6；Heterologous No. 2 of common wheat parent for hybridization and River wheat 41；Common wheat: China spring, good wheat No. 2, river educate 27, river agriculture 32, middle section wheat 169, Chongqing 98767, river wheat 30, river educate 31, River wheat 602, western section wheat 518, river wheat 36, river 12147, Mianyang wheat 318, river educate 17, Chuanmai 107, Nan Nong 02Y393, it is interior 2889, 18, another name for Sichuan Province wheat 921, river agriculture 17, river wheat 605, Zhou Mai 22, another name for Sichuan Province wheat 133, river wheat 58, river spoke are educated in inland river 24066, another name for Sichuan Province wheat 1602, river 14, river wheat 42, continuous wheat 285, continuous wheat 602, river wheat 604, river wheat 64, river wheat 104, river wheat 601, river wheat 1546, Hong Mai 161, silk floss 16, continuous wheat 51, river 07005, river wheat 1580 are educated in sun 335, another name for Sichuan Province wheat 969, river wheat 47, silk floss 06-458, river wheat 32, river agriculture 16, river.Phase Closing material can obtain from Triticeae Research Institute, Sichuan Agricultural University.Biochemical reagents used in the present invention are commercially available.

Each agent prescription of the present invention is as follows:

Protein extract buffer formula is as follows:

Prescription of its dyeing liquor is as follows:

Gel process for preparing is to use discontinuous buffer system, and it is as follows that separation gel buffer forms (pH 8.9):

Glue buffer (pH6.8): 1.0M Tris-HCl, 10% (W/V) SDS is concentrated.Buffer used in electrophoresis is dilution 10 Separation gel buffer again.

Polyacrylamide gel formula is as follows:

The acquisition of 1 primer pair of embodiment

The determination of ovum fringe goatweed 1Mx type glutelin sub-gene SNP marker and its detection primer, from NCBI data The complete encoding gene of ovum fringe goatweed 1Mx type HMW-GS is obtained in library, and gene order structure is analyzed.

Using the complete coded sequence of ovum fringe goatweed 1Mx high molecular weight subunit gene in ncbi database, (GenBank is stepped on Record number is KX375404.1) and other types (including common subunit in common wheat) high molecular weight subunit sequence includes: 1Dx2 (Genbank accession number: KF466259.1), 1Dy3 (Genbank accession number: KF466260.1), (Genbank is stepped on 1Ax Record number: JQ007589.1), 1Bx13 (Genbank accession number: EF540764.1), 1Dy11 (Genbank accession number: EU528008.1), 1By16 (Genbank accession number: EF540765.1), 1Ay (Genbank accession number: MF098417.1), 1Uy (Genbank accession number: KX375407.1), 1Ux (Genbank accession number: KX375406.1) gene order carry out sequence Analysis is compared, SNP site special in ovum fringe goatweed 1Mx type HMW-GS gene order is identified, has devised a pair of of ovum fringe The specific primer Ao-1Mx of goatweed 1Mx type subunit gene, nucleotide sequence are following (Fig. 1):

Upstream primer sequence: 5 '-CGCCCTCGTGGCTCTCACCC-3 '；

Downstream primer sequence: 5 ' TTTGCTGCTGGTATTGTCCA-3 '.

It is expected that the segment that the expected amplified production fragment length of PCR is 182bp.Molecular labeling of the invention can be used for identifying ovum Whether contain 1Mx type HMW-GS gene in fringe goatweed and wheat distance edge hybrid offspring, specific primer Ao-1Mx can be used for Molecular labeling of the PCR amplification to distinguish.

The foundation of ovum fringe goatweed 1Mx type HMW-GS gene tester in 2 wheat distance edge hybrid offspring of embodiment

1, the acquisition and identification of wheat distance edge hybrid offspring

It is female parent with ovum fringe goatweed AS6, artificial emasculation (stamen and bagging of little Hua in removing spikelet) is carried out before loose powder, Award that (clip just in the tassel of flowering, cuts off small ear, shakes anther with fresh heterologous No. 2 (YY2) pollen of common wheat after 3 days The female parent for falling into emasculation small is taken), then bagging obtains several seeds until harvest.The plant that the germination obtains continues As female parent, emasculation is pollinated using heterologous No. 2 (YY2) pollen of common wheat again, obtains a little seed.The seed of acquisition Sow the plant grown up to uses river wheat 41 (CM41) to hybridize again, later continuous selfing, obtains AS6/2/YY2//CM41F7 for seed.

Take every, seed of above-mentioned distant hybrid progeny it is with knife that its is crosscutting into two, take and ground containing albuminosus half granule seed Fine powder is worn into, is placed in the centrifuge tube of 1.5ml, adds 25 μ l protein extract buffers by every milligram of sample, is mixed, shaken at room temperature 1h is extracted, 6min is boiled in boiling water, 1200 r/min are centrifuged 6min, and supernatant is protein extraction sample.

When electrophoresis detection, 3 μ l point sample of protein extraction sample is drawn.SDS-PAGE electrophoresis is adjusted in constant current 20mA, voltage It is carried out under the conditions of 120V.1h is dyed with dyeing liquor after ten minutes within electrophoresis 2 hours, finally with water decolorization is distilled, shine after clear background Mutually save.

2, the acquisition and identification of ovum fringe goatweed AS6 and the filial generation seed of common wheat

The high-molecular-weight glutelin subunit in ovum fringe goatweed is reached into quality breeding to common wheat transfer to realize Purpose, according in step 1 method obtain ovum fringe goatweed AS6/2/YY2//CM41F7 generation, obtained seed is carried out Protein extraction, gel electrophoresis and observation, method is the same as above-mentioned steps 1；SDS-PAGE hybrid of the AS6/2/YY2//CM41F7 for seed Qualification result shows that the 1Mx type subunit of ovum fringe goatweed and common wheat parent's subunit have similar size, in backcross progeny seed In be likely to be obtained heredity, it is also possible to lose (Fig. 2).F7-1,2,3,4,5,6 indicate the seed in F7 generation obtained in Fig. 1, In, solid arrow indicates that the 1Mx type high-molecular-weight glutelin subunit for belonging to ovum fringe goatweed, dotted arrow indicate in common wheat There is the subunit of similar size (ovum fringe goatweed 1Mx type subunit or common wheat may be belonged to the 1Mx type subunit of ovum fringe goatweed Subunit or both common subunit presence).

There is the reason of foreign gene is lost and is that distant hybrid progeny chromosome is not sufficiently stable in offspring, in cell When division, it is easy to cause the variation or loss of certain character genes of hybrid parent, to cause these characters lacking in offspring It loses.Therefore, in order to continue tracing detection external source target gene in distant hybrid progeny, a set of gene specific molecular marker is developed Identifying exogenous genetic material from DNA level whether there is or not (true and false hybrid) is fast and efficiently method.Meanwhile as seen from Figure 2, (arrow in figure when having a gluten subunit of similar size for (1Mx type subunit) in ovum fringe goatweed AS6 and common wheat parent It is shown), it can not directly be distinguished from Wheat Background or external source ovum fringe goatweed by SDS-PAGE, to determine that it is accurate Source, it is necessary to by gene specific molecular marker.

3, extracting genome DNA

By the filial generation after SDS-PAGE is detected, ovum fringe goatweed AS6 and common wheat parent are planted in field, Listed number when plant strain growth is strain, takes young leaflet tablet to carry out extracting genome DNA, and extracting method uses CTAB method, mentions Take that steps are as follows:

A) the fresh young leaflet tablet of 1-2g is taken, is put into grinding alms bowl, appropriate liquid nitrogen is added and is rapidly ground to blade carefully, is put into In 50ml centrifuge tube, add isometric CTAB extracting solution (2%CTAB for being preheated to 65 DEG C；1.4M NaCl；0.1M Tris-HCl, PH=8.0；0.1M EDTA, pH=8.0；Use preceding plus 2% beta -mercaptoethanol) 15ml, it mixes.

B) 65 DEG C of water-bath 40min, jog mixes therebetween.

C) add isometric chloroform: isoamyl alcohol (24: 1, V:V) after being cooled to room temperature, mix gently to supernatant in milky white Color or milk shape, 4000r/min are centrifuged 10min.

D) supernatant is taken, adds isometric -20 DEG C of pre- cold isopropanols, is placed in ice bath and precipitates DNA.

E) choose DNA, washed 2 times with 70% alcohol, dehydrated alcohol is washed once, dry up DNA, be dissolved in appropriate pH=8.0 1 × In TE solution.

F) agarose gel electrophoresis detection DNA concentration and quality.

4, PCR specific amplification

PCR amplification is carried out using the genome of the above-mentioned each material of the primer pair of embodiment 1.

Ao-1Mx upstream primer sequence: 5 '-CGCCCTCGTGGCTCTCACCC-3 '

Ao-1Mx downstream primer sequence: 5 '-TTTGCTGCTGGTATTGTCCA-3 '.

PCR reaction system (25 μ l):

PCR response procedures: 94 DEG C of initial denaturation, 5min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 40s, totally 30 A circulation；Extend 72 DEG C of 10min eventually.

Ao-1Mx primer amplification fragment length is 182bp.Amplification is shown in Fig. 3, after amplified production is sequenced, on NCBI BLAST is carried out, shows that the segment of the 182bp of amplification belongs to ovum fringe goatweed 1Mx type high-molecular-weight glutelin subunit.In conjunction with Fig. 3 With BLAST as a result, illustrating that primer Ao-1Mx of the invention can specific amplification ovum fringe goatweed 1Mx type high-molecular-weight glutelin Asia Segment in base encoding gene, by compared with it can amplify target fragment in AS6, so that it is determined that external source ovum fringe goatweed 1Mx type subunit is present in AS6/2/YY2//CM41F7, and then true and false hybrid generation is distinguished.

5, the verifying of specific fragment

The recycling and purifying of PCR product are carried out using Ago-Gel DNA QIAquick Gel Extraction Kit (TIANGEN).Amplified production Carrier T is recycled and be connected to after purification, is carried out according to pMD19-T kit (TaKaRa) operation instructions, reaction solution is in 16 DEG C Water-bath connection is overnight.Competent escherichia coli cell is converted, well-grown white single colonie on the plate of picking overnight incubation, It crosses in the LB solid medium tablets containing ampicillin (50 μ g/mL), expands culture (37 DEG C of culture 5h).Picking weight Group bacterium carries out PCR amplification, and amplification uses Ao-1MX primer:

Ao-1Mx upstream primer sequence: 5 '-CGCCCTCGTGGCTCTCACCC-3 '；

Ao-1Mx downstream primer sequence: 5 '-TTTGCTGCTGGTATTGTCCA-3 '.

PCR reaction solution total volume is 25 μ l, wherein containing 2 × TaqPlus MasterMix (vazyme) 12.5 μ l, ddH₂O 10.5 μ l, Ao-1Mx upstream primer (10 μM), 1 μ l, Ao-1Mx downstream primer (10 μM), 1 μ l, a little hickie thallus.PCR reaction interval Sequence is same as above.After obtaining positive colony, Dalian treasured biotech firm is sent to be sequenced, sequencing result shows using Ao-1Mx primer rear The fragment sequence and ovum fringe goatweed 1Mx type glutelin sub-gene target area sequence expanded in generation is completely the same.Further Illustrate the method for 1Mx type glutelin sub-gene in remote with the wheat edge offspring of detection ovum fringe goatweed that the present invention establishes accurately, Reliably.

Embodiment 3 detects the specificity of 1Mx type HMW-GS gene primer in ovum fringe goatweed and wheat distance edge hybrid offspring Verifying

To ensure that the primer that the present invention is developed can really detect turning for ovum fringe goatweed 1Mx type HMW-GS gene It moves and exists, it is necessary to which the specificity of primer is verified.

According to the plant genome DNA extracting method and PCR amplification method in embodiment 2,46 wheat breeds are extracted (being) (China spring, good wheat No. 2, river educate 27, river agriculture 32,31, river wheat 602, west are educated in middle section wheat 169, Chongqing 98767, river wheat 30, river Section wheat 518, river wheat 36, river 12147, Mianyang wheat 318, river educate 17, Chuanmai 107, Nan Nong 02Y393, interior 2889, inland river 24066, Another name for Sichuan Province wheat 1602, river educate 18, another name for Sichuan Province wheat 921, river agriculture 17, river wheat 605, Zhou Mai 22, another name for Sichuan Province wheat 133, river wheat 58, river spoke 14, river wheat 42, Continuous wheat 285, continuous wheat 602, river wheat 604, river wheat 64, river wheat 104, river wheat 601, river wheat 1546, Hong Mai 161, Mianyang 335, another name for Sichuan Province wheat 969,16, continuous wheat 51, river 07005, river wheat 1580 are educated in river wheat 47, silk floss 06-458, river wheat 32, river agriculture 16, river, big by sichuan agriculture Wheat research institute is learned to save and provide) genomic DNA and carry out PCR augmentation detection, expansion using designed primer Ao-1Mx Increase detection method with embodiment 2.

Augmentation detection result is shown in Fig. 4, and as can be seen from Figure, primer of the invention fails to make in 46 productions for detection Purpose band is expanded in wheat, therefore is further demonstrated that, the detection ovum fringe goatweed high molecular weight 1Mx type paddy egg developed White subunit coding gene primer specificity is good, reaches 100% specifically, also illustrates ovum fringe goatweed high molecular weight 1Mx type paddy egg Bai Yaji is not imported into current wheat breed also, which can be applied in wheat breeding.Thus illustrate, utilize Primer provided by the invention can accurately track and detect to contain only ovum fringe goatweed high molecular weight 1Mx type gluten subunit base The wheat lines of cause, to be conducive to greatly improve wheat breeding efficiency.

It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and Modification, the scope of the present invention is defined by the appended.

Sequence table

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Claims (8)

1. a kind of molecular labeling for detecting ovum fringe goatweed and wheat hybridizing HMW-GS gene according to claim 1, It is characterized in that, the molecular labeling is obtained by primer pair Ao-1Mx through PCR amplification, the nucleotide of the primer pair Ao-1Mx Sequence is as shown in NO.1~2 SEQ ID.

2. a kind of primer for detecting ovum fringe goatweed and wheat hybridizing HMW-GS gene, which is characterized in that the primer is Ao- 1Mx。

3. a kind of method for detecting ovum fringe goatweed and wheat hybridizing HMW-GS gene, which comprises the following steps: use Primer Ao-1Mx carries out PCR method and expands hybrid wheat genomic DNA to be checked, if can be amplified with primer Ao-1Mx The amplified fragments of 182bp, then illustrating the wheat to be checked, there are ovum fringe goatweed 1Mx type glutelin sub-genes.

4. a kind of method for detecting ovum fringe goatweed and wheat hybridizing HMW-GS gene according to claim 3, feature It is, pcr amplification reaction program are as follows: 94 DEG C of initial denaturation, 5min；94 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 40s, altogether 30 circulations；Extend 72 DEG C of 10min eventually.

5. a kind of kit, which is characterized in that the kit contains primer Ao-1Mx.

6. a kind of kit according to claim 5, which is characterized in that above-mentioned kit also contains positive control mould Plate, the positive control template are the plasmid containing ovum fringe goatweed 1Mx type HMW-GS encoding gene.

7. kit described in molecular labeling described in claim 1 or primer as claimed in claim 2 or claim 5 is being examined Survey the application of ovum fringe goatweed 1Mx type HMW-GS gene in wheat distance edge hybrid hybrid.

8. kit described in molecular labeling described in claim 1 or primer as claimed in claim 2 or claim 5 is small Application in wheat filial generation breeding.