CN106011299A - Specific molecular markers for detection of Aegilops comosa 2M, 3M, 6M and 7M chromosomes in wheat, kit and method - Google Patents
Specific molecular markers for detection of Aegilops comosa 2M, 3M, 6M and 7M chromosomes in wheat, kit and method Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to specific molecular markers for detection of Aegilops comosa 2M, 3M, 6M and 7M chromosomes in wheat, and the specific molecular markers are DNA base sequences respectively shown as SEQ ID No.1 and -18 in the sequence table. A to-be-detected chromosome sample and a specific molecular marker composition are put into a kit to conduct amplification. The specific molecular markers and the kit can be used for detection or assisted detection of the existence of Aegilops comosa 2M, 3M, 6M and 7M chromosomes in wheat. The invention provides an effective detection means for screening and identification of wheat-Aegilops comosa distant hybridization new germplasms involving Aegilops comosa 2M, 3M, 6M and 7M chromosomes, provides a detection method for transfer of excellent genes on Aegilops comosa 2M, 3M, 6M and 7M chromosomes to wheat by means of chromosome segments, and has positive industrialization value.
Description
Technical field
The present invention relates to biology field, particularly to Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi
Specific molecular marker, further relate to the test kit containing this specific molecular marker, and Aegilops comosa 2M in detection Semen Tritici aestivi, 3M,
The method of 6M and 7M chromosome.
Background technology
Aegilops comosa (Aegilops comosa, 2n=2x=14, genome MM), high Stripe rust resistance, leaf rust, stalk
The multiple diseases such as rust and powdery mildew (Friebe B, Jiang JM, Raupp WJ, et al.Characterization of wheat-alien
translocations conferring resistance to diseases and pests:current status.Euphytica,1996,
91:59-87；Gill BS,Sharma HC,Raupp WJ,et al.Evaluation of Aegilops species for resistance to
Wheat powdery mildew, wheat leaf rust, Hessian fly and greenbug.Plant Dis, 1985,69:314-316), it is
The favorable genes source of wheat breeding.From eighties of last century sixties, the Riley seminar of Britain, Miller seminar and China
Dong Yu treasure seminar carried out the distant hybridization work of Aegilops comosa and Semen Tritici aestivi respectively, in addition, have no other system
Carry out the research of Semen Tritici aestivi-Aegilops comosa distant hybridization work.The transfer of Aegilops comosa kind matter is all given little by above three seminar
Wheat, obtains a collection of Semen Tritici aestivi-Aegilops comosa distant hybridization germ plasm resource respectively.
After obtaining wheat distance edge hybrid germ plasm resource, it is dye to its accurate identification from the levels such as molecule, cell and biochemistry
The very important previous work of colour solid Engineering Breeding.In Semen Tritici aestivi in terms of Aegilops comosa Chromosome Identification, Friebe etc. establishes top
Awns goatweed chromosome standard C divides band, however the Aegilops comosa chromosome banding of separate sources the most variant Friebe etc.,
Standard karyotypes of Aegilops uniaristata,Ae.mutica,Ae.comosa subspecies comosa and
heldreichii(Poaceae).Plant Systematics and Evolution,1996,202:199-210}.Nasuda etc. and father-in-law's leap etc.
Set up respectively Aegilops comosa 2M chromosome, 1M-7M chromosome restricted length polymorphism (RFLP) labelling Nasuda etc.,
Structural rearrangement in chromosome 2M of Aegilops comosa has prevented the utilization of
the Compair and related wheat-Ae.comosa translocations in wheat improvement.Theor Appl
Genet,1998,96:780-785.Father-in-law's leaps etc., utilize RFLP molecular markers for identification Semen Tritici aestivi-Aegilops comosa alien substitution, heredity
Journal, 1997,24 (3): 248-254.Father-in-law's leap etc., Semen Tritici aestivi M genomic RFLP labelling, Journal of Agricultural Biotechnology,
1997,5(3):211-215)}.Molna ' r etc. establish Aegilops comosa chromosome flow cytometer method for sorting Molna ' r etc.
Chromosome Isolation by flow sorting in Aegilops umbellulata and Ae.comosa and their
allotetraploid hybrids Ae.biuncialis and Ae.geniculata.PLoS ONE,2011,6(11):e27708}。
In the method for above-mentioned qualification Aegilops comosa chromosome, chromosome C divides band operation sequence to waste time and energy, and Jim Sa used contaminates
Liquid quality, treatment temperature are different from time etc. easily causes result poor repeatability；RFLP relate to radioelement use and time-consuming the most relatively
Long；Flow cytometer costliness and complex operation step；Thus, set up wide spectrum and the letter of Aegilops comosa chromosome in detection Semen Tritici aestivi
Easily experimental technique is highly desirable to.
In view of the deficiency of aforementioned research, we establish Aegilops comosa chromosome cytogenetic markers (national inventing patent Shen
Please number: 201610301879.2, in substantive examination), Aegilops comosa in Semen Tritici aestivi can be dyeed from cytogenetics level by the method
Body is effectively identified.But, the authentication method of molecular biology level lacks the most very much.
Summary of the invention
In order to solve the problem not having Aegilops comosa chromosome PCR labelling in currently available technology, the invention provides a kind of energy
Reach and identify the spy whether containing Aegilops comosa 2M, 3M, 6M and 7M chromosome in sample to be tested the most fast, effectively, easily
Different molecular marker.
Present invention also provides the test kit containing above-mentioned specific molecular marker.
It is a further object to provide the molecule inspection of Aegilops comosa 2M, 3M, 6M and 7M chromosome in a kind of Semen Tritici aestivi
Survey method, is applied to screening and the appraisal of Semen Tritici aestivi-Aegilops comosa filial generation by the molecular marker of foundation.
The present invention is obtained through the following steps:
Our previous experiments finds, Aegilops comosa 2M and 7M chromosome contain Stripe rust resistance and powdery mildew respectively
New gene, 3M and 6M chromosome can significantly improve wheat tillering power and mass of 1000 kernel after importing Semen Tritici aestivi under specific ecological environment respectively,
Therefore, the present invention establishes from molecular level and can effectively identify Aegilops comosa in Semen Tritici aestivi (2M, 3M, 6M and 7M)
The authentication method of chromosome, to the Semen Tritici aestivi-Aegilops comosa hybridization relating to Aegilops comosa 2M, 3M, 6M and 7M chromosome
The qualification result of offspring shows, the molecular detecting method that this research is set up can be widely applied to relate to Aegilops comosa 2M, 3M,
The screening of the Semen Tritici aestivi of 6M and 7M chromosome-Aegilops comosa breeding new germ plasm and appraisal.
(1) with Aegilops comosa, China spring-Aegilops comosa amphidiploid, China spring, wheat breed Jimai 22 and Jinan 17
For material, design and screen chromosome second and third, six and seven homology group's PLUG primers.Therefrom screening can be at Aegilops comosa
(China spring, wheat breed Jimai is compared with the primer amplifying additional polymorphism band in China spring-Aegilops comosa amphidiploid
22 and Jinan 17).
(2) with the above-mentioned primer filtered out, China spring-Aegilops comosa amphidiploid, China spring, China spring-ovum fringe mountain are expanded
Leymus chinensis (Trin.) Tzvel. 1MgAddition line, China spring-Aegilops comosa 2M addition line, China spring-Aegilops comosa 3M addition line, China spring-
Aegilops comosa 4M addition line, China spring-Aegilops comosa 5M addition line, China spring-Aegilops comosa 6M addition line, in
State's spring-Aegilops comosa 6M (6A) substitution line and China spring-Aegilops comosa 7M addition line, obtained above-mentioned from tip awn goat
The additional polymorphism band (comparing China spring, wheat breed Jimai 22 and Jinan 17) of grass is positioned at 1M-7M or a few
On bar chromosome.
(3) with the above-mentioned primer filtered out, corresponding addition line/wheat hybridizing offspring is expanded, checking polymorphism mark
Application feasibility.
The specific molecular marker of Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi, for Aegilops comosa 2M,
The specific molecular marker compositions of the specific molecular marker composition of 3M, 6M and 7M chromosome, wherein Aegilops comosa 2M dye
The specific molecular marker of colour solid is CD913720-PLUG or CK171581-PLUG；Aegilops comosa 3M chromosome special
Molecular marker is CD491237-PLUG or CK207576-PLUG；The specific molecular marker of Aegilops comosa 6M chromosome is
CK168221-PLUG or CK206466-PLUG；The specific molecular marker of Aegilops comosa 7M chromosome is
CK214770-PLUG or CK203272-PLUG；
Described labelling CD913720-PLUG correspondence primer is by two lists shown in SEQ ID No.1 in sequence table and SEQ ID No.2
Chain DNA forms；
Described labelling CK171581-PLUG correspondence primer is by two lists shown in SEQ ID No.3 in sequence table and SEQ ID No.4
Chain DNA forms；
Described labelling CD491237-PLUG correspondence primer is by two lists shown in SEQ ID No.5 in sequence table and SEQ ID No.6
Chain DNA forms；
Described labelling CK207576-PLUG correspondence primer is by two lists shown in SEQ ID No.7 in sequence table and SEQ ID No.8
Chain DNA forms；
Described labelling CK168221-PLUG correspondence primer is by two lists shown in SEQ ID No.9 in sequence table and SEQ ID No.10
Chain DNA forms；
Described labelling CK206466-PLUG correspondence primer is by two lists shown in SEQ ID No.11 in sequence table and SEQ ID No.12
Chain DNA forms；
Described labelling CK214770-PLUG correspondence primer is by two shown in SEQ ID No.13 in sequence table and SEQ ID No.14
Single stranded DNA forms；
Described labelling CK203272-PLUG correspondence primer is by two shown in SEQ ID No.15 in sequence table and SEQ ID No.16
Single stranded DNA forms.
The test kit of Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi, containing above-mentioned Aegilops comosa 2M,
The specific molecular marker compositions of the specific molecular marker composition of 3M, 6M and 7M chromosome.
The method of Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi, by chromosome sample to be checked with special point
Sub-marking composition or put in test kit expands, and amplified production uses electrophoresis detection.
If containing 900bp or 660bp polymorphic bands in amplified production, containing Aegilops comosa 2M in chromosome sample the most to be checked
Chromosome；
If containing 780bp or 1700bp polymorphic bands in amplified production, containing Aegilops comosa 3M in chromosome sample the most to be checked
Chromosome；
If containing 710bp or 650bp polymorphic bands in amplified production, containing Aegilops comosa 6M in chromosome sample the most to be checked
Chromosome；
If containing 1200bp or 770bp polymorphic bands in amplified production, containing Aegilops comosa 7M in chromosome sample the most to be checked
Chromosome.
Preferably amplification reaction system is 15 μ L, including sample DNA 1 μ L, the 5U/ μ L Taq DNA of 25ng/ μ L
Polymerase 0.15 μ L, the dNTPs of 200 μMs, containing Mg2+10 × PCR buffer 1.5 μ L, the upstream and downstream of 10 μMs
The each 1 μ L of primer, supplements reaction system to 15 μ L with aseptic DDW.
The preferably ratio of the specific molecular marker of 2M, 3M, 6M and 7M chromosome is 1:1:1:1.
Preferably amplification program is: 94 DEG C of denaturations 3min, subsequently 35 circulations: 94 DEG C of degeneration 45S, 57 DEG C of annealing 45S,
72 DEG C extend 2min, and last 72 DEG C extend 10min, 4 DEG C of preservations.
Described specific molecular marker or the application in wheat breeding of the described test kit.
Described specific molecular marker or described test kit detection or auxiliary detection Semen Tritici aestivi whether contain Aegilops comosa 2M,
Application in 3M, 6M and 7M chromosome.
Beneficial effects of the present invention:
(1) present invention establishes Aegilops comosa 2M, 3M, 6M and 7M chromosomal marker, it is provided that push up in detection Wheat Background
The new method of awns goatweed 2M, 3M, 6M and 7M chromosome；
(2) the chromosome specific molecular marker that the present invention sets up, to relating to Aegilops comosa 2M, 3M, 6M and 7M chromosome
The screening of Semen Tritici aestivi-Aegilops comosa distant hybridization new germ plasm and qualification provide effective detection means；
(3) the chromosome specific molecular marker that the present invention sets up, shifts Aegilops comosa in the way of chromosome segment to Semen Tritici aestivi
Favorable genes (such as stripe rust of wheat and powdery mildew disease-resistant gene etc.) on 2M, 3M, 6M and 7M chromosome provides
Detection method, has a positive industrialization value.
Accompanying drawing explanation
Fig. 1. primer CD913720 (A), CK171581 (B), CD491237 (C), CK207576 (D), CK168221
(E), CK206466 (F), CK214770 (G) and the amplification of CK203272 (H)；
Note: M is Marker, and molecular size range is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp,
In A-H, swimming lane 1-5 represents Aegilops comosa, China spring-Aegilops comosa amphidiploid, China spring, wheat breed respectively
Jimai 22 and Jinan 17；
Fig. 2. primer CD913720 (A), CK171581 (B), CD491237 (C), CK207576 (D), CK168221
(E), CK206466 (F), CK214770 (G) and the amplification of CK203272 (H)；
Note: M is Marker, and molecular size range is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp,
In A-H, swimming lane 1-10 represents China spring-Aegilops comosa amphidiploid, China spring, China spring-ovum fringe goatweed 1M respectivelygAttached
Add be, China spring-Aegilops comosa 2M addition line, China spring-Aegilops comosa 3M addition line, China spring-Aegilops comosa
4M addition line, China spring-Aegilops comosa 5M addition line, China spring-Aegilops comosa 6M addition line, China spring-tip awn mountain
Leymus chinensis (Trin.) Tzvel. 6M (6A) substitution line and China spring-Aegilops comosa 7M addition line；
Fig. 3. primer CD913720 (A), CK171581 (B), CD491237 (C), CK207576 (D), CK168221
(E), CK206466 (F), CK214770 (G) and the amplification of CK203272 (H)；
Note: M is Marker, and molecular size range is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp,
In A, swimming lane 1-24 represents Aegilops comosa, China spring-Aegilops comosa 2M addition line, China spring, China spring-tip awn respectively
Goatweed 2M addition line/China spring filial generation P1-P21；In B, swimming lane 1-24 represents Aegilops comosa, China spring-top respectively
Awns goatweed 2M addition line, China spring, China spring-Aegilops comosa 3M addition line/China spring filial generation P22-P42；C
Middle swimming lane 1-24 represents Aegilops comosa, China spring-Aegilops comosa 3M addition line, China spring, China spring-tip awn mountain respectively
Leymus chinensis (Trin.) Tzvel. 3M addition line/China spring filial generation Q1-Q21；In D, swimming lane 1-24 represents Aegilops comosa, China spring-tip awn respectively
Goatweed 3M addition line, China spring, China spring-Aegilops comosa 2M addition line/China spring filial generation Q22-Q42；In E
Swimming lane 1-24 represents Aegilops comosa, China spring-Aegilops comosa 6M addition line, China spring, China spring-tip awn goat respectively
Grass 6M addition line/China spring filial generation R1-R21；In F, swimming lane 1-24 represents Aegilops comosa, China spring-tip awn mountain respectively
Leymus chinensis (Trin.) Tzvel. 6M addition line, China spring, China spring-Aegilops comosa 6M addition line/China spring filial generation R22-R42；G swims
Road 1-24 represents Aegilops comosa, China spring-Aegilops comosa 7M addition line, China spring, China spring-Aegilops comosa respectively
7M addition line/China spring filial generation S1-S21；In H, swimming lane 1-24 represents Aegilops comosa, China spring-tip awn goat respectively
Grass 7M addition line, China spring, China spring-Aegilops comosa 7M addition line/China spring filial generation S22-S42.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described:
Experiment material
PLUG primer used is submitted to according to Semen Tritici aestivi EST location Engineering Network (http://wheat.pw.usda.gov/NSF/data.html)
Est sequence design, primer synthesis completed by Chengdu Rui Xin biotech firm.
Aegilops comosa (TA1967) and China spring-ovum fringe goatweed 1MgAddition line (TA7655) is state by kansas, U.S.A
University's Wheat volatiles provides with genetic resources center Raupp doctor J, and the public can lose from kansas, U.S.A state university Semen Tritici aestivi
Passing and obtain with compensation with genome resource center (http://www.k-state.edu/wgrc/), this biomaterial only attaches most importance to what duplicate was invented
Used by related experiment, do not make other purposes and use.Semen Tritici aestivi China spring (CS) is by Life Science and Technology institute of University of Electronic Science and Technology
Professor Li Guangrong provides.Wheat breed Jinan 17 and Jimai 22 are independently bred as by patent applicant place wheat genetic breeding team.
China spring, Jinan 17 and the seed of Jimai 22, the public can obtain from Crop Inst. of shandong Prov. Agriculture science Academy, this biology material
Material only attach most importance to duplicate invention related experiment used by, can not use as other purposes.China spring-Aegilops comosa amphidiploid
(XX039) it is that China spring hybridizes with Aegilops comosa, hybrid F1Through doubling acquisition, the public can make from Shandong Academy of Agricultural Sciences
Thing institute obtain, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes.China
Spring-Aegilops comosa 2M addition line, China spring-Aegilops comosa 3M addition line, China spring-Aegilops comosa 4M addition line,
China spring-Aegilops comosa 5M addition line, China spring-Aegilops comosa 6M addition line, China spring-Aegilops comosa 6M (6A)
Substitution line and China spring-Aegilops comosa 7M addition line are provided by Reader professor SM at Britain John Ying Nasi center, the public
Gratuitously can obtain from Britain John Ying Nasi center (https: //www.jic.ac.uk/germplasm/), this biomaterial is only for repeating
Used by the related experiment of the present invention, can not use as other purposes.P1-P41 be China spring-Aegilops comosa 2M addition line/
China spring hybridization F2Progeny material；Q1-Q41 is China spring-Aegilops comosa 3M addition line/China spring hybridization F2Progeny material；
R1-R41 is China spring-Aegilops comosa 6M addition line/China spring hybridization F2Progeny material；S1-S41 is China spring-tip awn mountain
Leymus chinensis (Trin.) Tzvel. 7M addition line/China spring hybridization F2Progeny material.
Experimental technique
PLUG primer amplification reaction system is 15 μ L, including template DNA 1 μ L, the 5U/ μ L Taq DNA of 25ng/ μ L
Polymerase (Shen Neng betting office) 0.15 μ L, the dNTPs (Bo Ao company) of 200 μMs, containing Mg2+10 × PCR buffer
1.5 μ L (Shen Neng betting office), each 1 μ L of upstream and downstream primer of 10 μMs, supplement reaction system extremely with aseptic DDW
15μL。
PCR reaction is carried out in BIO-RAD PCR amplification instrument, and amplification program is: 94 DEG C of denaturations 3min, 35 subsequently
Circulation: 94 DEG C of degeneration 45S, 57 DEG C of annealing 45S, 72 DEG C extend 2min, and last 72 DEG C extend 10min, 4 DEG C of preservations.
Amplified production carries out electrophoresis on the agar gel of 2%, and electrophoretic buffer is 1 × TAE.Amplified production 10ul is under 150V constant voltage
Electrophoresis about 25min, then carries out the 30min that dyes, finally at GDS-Gel Dol 2000 with the ethidium bromide solution of 1ug/mL
Under ultraviolet gel imaging system, scanning takes pictures.
Embodiment 1
With Aegilops comosa, China spring-Aegilops comosa amphidiploid, China spring, wheat breed Jimai 22 and Jinan 17 as material
Material, design and screen chromosome second and third, six and seven homology group's PLUG primer 41 to, 84 to, 53 to and 73 right, expand
Volume increase thing carries out enzyme action with restricted enzyme HaeIII and TaqI, by the pcr amplification product from same primer (not respectively
Enzyme action, HaeIII enzyme action and three kinds of situations of TaqI enzyme action) carry out agarose gel electrophoresis, found that chromosome the second homology group
CD913720 (Figure 1A) and CK171581 (Figure 1B), the CD491237 (Fig. 1 C) of chromosome the 3rd homology group and
CK207576 (Fig. 1 D), the CK168221 (Fig. 1 E) and CK206466 (Fig. 1 F) of chromosome the 6th homology group, dyeing
The CK214770 (Fig. 1 G) and CK203272 (Fig. 1 H) of body the 7th homology group can be on Aegilops comosa and China spring-tip awn mountains
Leymus chinensis (Trin.) Tzvel. amphidiploid amplifies additional polymorphism band, these polymorphic bands sizes be respectively 900bp, 660bp, 780bp,
1700bp, 710bp, 650bp, 1200bp and 770bp (in Figure 1A-H shown in arrow), but, Semen Tritici aestivi comparison China spring,
Jimai 22 and Jinan 17 all can not expand these bands, illustrate that these polymorphic bandses are from Aegilops comosa.Above-mentioned 8 to drawing
The information such as the sequence of thing, chromosome of wheat position, place and polymorphism mark length are shown in Table 1.
Table 1. Aegilops comosa chromosome specific molecular marker information
Embodiment 2
So far, owing to there are no the report that China spring-Aegilops comosa 1M addition line is formulated, and Aegilops comosa M dye
Colour solid is the M of ovum fringe goatweedgThe donor of chromosome, may have occurred chromosomal section in species polyploidization and evolutionary process
Restructuring or variation, cause the chromosome coming from Aegilops comosa in ovum fringe goatweed slightly different with Aegilops comosa M chromosome,
Therefore, M is usedgRepresent to show difference.Therefore, in the case of there is no China spring-Aegilops comosa 1M addition line, Ke Yiyong
China spring-ovum fringe goatweed 1MgAddition line makes marks location.
In order to the above-mentioned polymorphic bands from Aegilops comosa is positioned at certain in Aegilops comosa 1M-7M or a few
On chromosome, with the above-mentioned 8 pairs of PLUG primers filtered out to China spring-Aegilops comosa amphidiploid, China spring, China spring
-ovum fringe goatweed 1MgAddition line, China spring-Aegilops comosa 2M addition line, China spring-Aegilops comosa 3M addition line,
China spring-Aegilops comosa 4M addition line, China spring-Aegilops comosa 5M addition line, China spring-Aegilops comosa 6M are attached
Add be, China spring-Aegilops comosa 6M (6A) substitution line and China spring-Aegilops comosa 7M addition line expand, obtain respectively
Following result:
Primer CD913720 and CK171581 can be at China spring-Aegilops comosa amphidiploid and China spring-Aegilops comosa
2M addition line amplifies molecular weight respectively and is about 900bp (Fig. 2 A) and the specific polymorphism band of 660bp (Fig. 2 B),
But, China spring and other 6 addition line (1Mg, 3M-7M) do not amplify respective strap, this two polymorphism bars are described
Band is Aegilops comosa 2M chromosome specific molecular marker, is designated as CD913720-PLUG and CK171581-PLUG (table 1).
Primer CD491237 and CK207576 can be at China spring-Aegilops comosa amphidiploid and China spring-Aegilops comosa
3M addition line amplifies molecular weight respectively and is about 780bp (Fig. 2 C) and the specific polymorphism band of 1700bp (Fig. 2 D),
But, China spring and other 6 addition line (1Mg, 2M, 4M-7M) do not amplify respective strap, illustrate these two polymorphic
Property band is Aegilops comosa 3M chromosome specific molecular marker, is designated as CD491237-PLUG and CK207576-PLUG (table
1)。
Primer CK168221 and CK206466 can be at China spring-Aegilops comosa amphidiploid and China spring-Aegilops comosa
6M addition line amplifies molecular weight respectively and is about 710bp (Fig. 2 E) and the specific polymorphism band of 650bp (Fig. 2 F),
But, China spring and other 6 addition line (1Mg, 3M-5M and 7M) do not amplify respective strap, illustrate that these are more than two
State property band is Aegilops comosa 6M chromosome specific molecular marker, is designated as CK168221-PLUG and CK206466-PLUG
(table 1).
Primer CK214770 and CK203272 can be at China spring-Aegilops comosa amphidiploid and China spring-Aegilops comosa
7M addition line amplifies molecular weight respectively and is about 1200bp (Fig. 2 G) and the specific polymorphism band of 770bp (Fig. 2 H),
But, China spring and other 6 addition line (1Mg, 3M-6M) do not amplify respective strap, this two polymorphism bars are described
Band is Aegilops comosa 7M chromosome specific molecular marker, is designated as CK214770-PLUG and CK203272-PLUG (table 1).
Embodiment 3
In order to verify the practicality of above-mentioned 8 labellings, respectively 4 colonies are expanded with the above-mentioned 8 pairs of primers filtered out,
The practicality of verification mark.
Wherein, with CD913720 and CK171581 of chromosome the second homology group to China spring-Aegilops comosa 2M addition line/
China spring hybridization F2 progeny material P1-P42 is expanded, and result shows, CD913720 and CK171581 all can be at Semen Tritici aestivi
-Aegilops comosa amphidiploid, Semen Tritici aestivi-Aegilops comosa 2M addition line, P2-P6, P16-P20, P25, P26, P28, P31,
P32, P36, P39, P40 and P42 amplify polymorphism mark CD913720-PLUG and CK171581-PLUG, and
China spring and P1, P7-P15, P21-P24, P27, P29, P30, P33-P35, P37, P38 and P41 do not amplify
Corresponding polymorphic bands (table 2).Wherein, CD913720 to the amplification of P1-P21 as shown in Figure 3A, CK171581
To the amplification of P22-P42 as shown in Figure 3 B.
With CD491237 and CK207576 of chromosome the 3rd homology group to China spring-Aegilops comosa 3M addition line/China spring
Hybridization F2 progeny material Q1-Q41 is expanded, and result shows, CD491237 and CK207576 all can be at Semen Tritici aestivi-tip awn
Goatweed amphidiploid, Semen Tritici aestivi-Aegilops comosa 3M addition line, Q1, Q4-Q6, Q11, Q12, Q14, Q15, Q20,
Q21, Q23-Q27, Q31, Q33, Q34 and Q38-40 amplify polymorphism mark CD491237-PLUG and
CK207576-PLUG, and China spring and Q2, Q3, Q7-Q10, Q13, Q16-Q19, Q22, Q28-30, Q32,
Q35-37, Q41 and Q42 do not amplify corresponding polymorphic bands (table 3).Wherein, CD491237 is to Q1-Q21's
As shown in Figure 3 C, CK207576 is to the amplification of Q22-Q42 as shown in Figure 3 D for amplification.
With CK168221 and CK206466 of chromosome the 6th homology group to China spring-Aegilops comosa 6M addition line/China spring
Hybridization F2 progeny material R1-R41 is expanded, and result shows, CK168221 and CK206466 all can be at Semen Tritici aestivi-tip awn
Goatweed amphidiploid, Semen Tritici aestivi-Aegilops comosa 6M addition line, R1, R2, R5, R6, R8, R13, R14, R19,
R20, R27-R29, R31, R35-R37 and R41 amplify polymorphism mark CK168221-PLUG and CK206466-PLUG,
And at China spring and R3, R4, R7, R9-R12, R15-R18, R21-R26, R30, R32-R34, R38-R40 and R42
In do not amplify corresponding polymorphic bands (table 4).Wherein, CK168221 to the amplification of R1-R21 as shown in FIGURE 3 E,
CK206466 is to the amplification of R22-R42 as illustrated in Figure 3 F.
With CK214770 and CK203272 of chromosome the 7th homology group to China spring-Aegilops comosa 7M addition line/China spring
Hybridization F2 progeny material S1-S41 is expanded, and result shows, CK214770 and CK203272 all can be at Semen Tritici aestivi-tip awn
Goatweed amphidiploid, Semen Tritici aestivi-Aegilops comosa 7M addition line, S1-S3, S8, S10-S15, S18, S19, S22, S23,
S25, S28, S30 and S37-S40 amplify polymorphism mark CK214770-PLUG and CK203272-PLUG, and in
State's spring and S4-S7, S9, S16, S17, S20, S21, S24, S26, S27, S29, S31-S36, S41 and S42 do not have
Amplify corresponding polymorphic bands (table 5).Wherein, CK214770 to the amplification of S1-S21 as shown in Figure 3 G,
CK203272 is to the amplification of S22-S42 as shown in figure 3h.
Above primer all can amplify corresponding molecular marker in corresponding filial generation material, and the chromosome that the present invention sets up is described
Specific molecular marker can relate to the filial generation of 2M, 3M, 6M and 7M chromosome to Semen Tritici aestivi-Aegilops comosa to be carried out effectively
Screening and Preliminary Identification, therefore, have practical value in Screening of Germplasm with wheat breeding.
Table 2. Aegilops comosa 2M chromosome specific molecular marker CD913720-PLUG, CK171581-PLUG are to filial generation colony
Molecular Detection situation
Table 3. Aegilops comosa 3M chromosome specific molecular marker CD491237-PLUG, CK207576-PLUG are to filial generation colony
Molecular Detection situation
Table 4. Aegilops comosa 6M chromosome specific molecular marker CK168221-PLUG, CK206466-PLUG are to filial generation colony
Molecular Detection situation
Table 5. Aegilops comosa 7M chromosome specific molecular marker CK214770-PLUG, CK203272-PLUG are to filial generation colony
Molecular Detection situation
In above-described embodiment 1,2,3, the specific molecular marker for Aegilops comosa 2M, 3M, 6M and 7M chromosome enters respectively
Go test, it was demonstrated that specific molecular marker may be used for differentiating Aegilops comosa 2M, 3M, 6M and 7M chromosome.And
The purpose of the present invention, is to provide the compositions of the specific molecular marker of a kind of Aegilops comosa 2M, 3M, 6M and 7M chromosome,
Aegilops comosa 2M, 3M, 6M and 7M chromosome in measuring samples can be detected by this compositions simultaneously, and from
The content that above-described embodiment 1,2,3 is recorded it can be seen that will not interfere between testing result, detection quickly, convenient,
Accurately, breeding work is had very useful promotional value.Judge to make amplification procedure, result be more prone to, tip awn mountain
The mol ratio of the specific molecular marker of Leymus chinensis (Trin.) Tzvel. 2M, 3M, 6M and 7M chromosome is 1:1:1:1.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention should not be limited by the examples, other
The change made under any spirit without departing from the present invention and principle, modify, combine, substitute, simplify and all should be equivalence and replace
Change mode, within being included in protection scope of the present invention.
<110>Crop Inst. of shandong Prov. Agriculture science Academy
<120>specific molecular marker, test kit and the method for Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi
<160> 16
<210> 1
<211>20
<212>
DNA
<213>synthetic
<400> 1
ACTCGCGTGT ATTTGCTTCC 20
<210> 2
<211> 22
<212>
DNA
<213>synthetic
<400> 2
AATGATGAAA
CCCTTCTTCC TG 22
<210> 3
<211> 20
<212>
DNA
<213>synthetic
<400> 3
GACCGGTGCA TCTTAAGGAA 20
<210> 4
<211> 20
<212>
DNA
<213>synthetic
<400> 4
AGCCTGAAGG
AATGGTTGAG 20
<210> 5
<211> 20
<212>
DNA
<213>synthetic
<400> 5
CCAACCTCTT
GGAGCACATT 20
<210> 6
<211> 20
<212>
DNA
<213>synthetic
<400> 6
CAGCAGTCAC
AGCACAACCT 20
<210> 7
<211> 20
<212>
DNA
<213>synthetic
<400> 7
AACAAATTCC
GCATCTGGTC 20
<210> 8
<211> 20
<212>
DNA
<213>synthetic
<400> 8
TTCGTGATGA
ACGCGTATGT 20
<210> 9
<211> 20
<212>
DNA
<213>synthetic
<400> 9
TCTTGCCATA
TCCTCCCATC 20
<210> 10
<211> 20
<212>
DNA
<213>synthetic
<400> 10
AGCTAAAGCC
ACTCCGACAA 20
<210> 11
<211> 20
<212>
DNA
<213>synthetic
<400> 11
GTGAGTTAAA
AGCCCGACCA 20
<210> 12
<211> 20
<212>
DNA
<213>synthetic
<400>12
AGACTCGCTG
GATGCAGATT 20
<210> 13
<211> 20
<212>
DNA
<213>synthetic
<400>13
CGGTGAACCC
CACTGTAAAT 20
<210>14
<211> 20
<212>
DNA
<213>synthetic
<400> 14
ACGTACTTCG
GGCTCTTCCT 20
<210> 15
<211> 21
<212>
DNA
<213>synthetic
<400> 15
TGTGGAGCAG
TGCTGTTTAT G 21
<210> 16
<211> 20
<212>
DNA
<213>synthetic
<400> 16
GGTACGGGGA
CAACTCACAG 20
Claims (9)
1. the specific molecular marker of Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi, the specific molecular marker compositions formed for the specific molecular marker of Aegilops comosa 2M, 3M, 6M and 7M chromosome, it is characterised in that the specific molecular marker of Aegilops comosa 2M chromosome is CD913720-PLUG or CK171581-PLUG；The specific molecular marker of Aegilops comosa 3M chromosome is CD491237-PLUG or CK207576-PLUG；The specific molecular marker of Aegilops comosa 6M chromosome is CK168221-PLUG or CK206466-PLUG；The specific molecular marker of Aegilops comosa 7M chromosome is CK214770-PLUG or CK203272-PLUG；
Described labelling CD913720-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.1 and SEQ ID No.2；
Described labelling CK171581-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.3 and SEQ ID No.4；
Described labelling CD491237-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.5 and SEQ ID No.6；
Described labelling CK207576-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.7 and SEQ ID No.8；
Described labelling CK168221-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.9 and SEQ ID No.10；
Described labelling CK206466-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.11 and SEQ ID No.12；
Described labelling CK214770-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.13 and SEQ ID No.14；
Described labelling CK203272-PLUG correspondence primer is by SEQ in sequence table
Two single stranded DNA compositions shown in ID No.15 and SEQ ID No.16.
2. the test kit of Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi, it is characterised in that the specific molecular marker compositions of the specific molecular marker composition containing Aegilops comosa 2M, 3M, 6M and 7M chromosome in the claims 1.
3. the method for Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection Semen Tritici aestivi, it is characterized in that to expand in chromosome sample to be checked and the specific molecular marker compositions in claim 1 or the test kit putting in claim 2, amplified production uses electrophoresis detection.
Method the most according to claim 3, it is characterised in that
If containing 900bp or 660bp polymorphic bands in amplified production, containing Aegilops comosa 2M chromosome in chromosome sample the most to be checked；
If containing 780bp or 1700bp polymorphic bands in amplified production, containing Aegilops comosa 3M chromosome in chromosome sample the most to be checked；
If containing 710bp or 650bp polymorphic bands in amplified production, containing Aegilops comosa 6M chromosome in chromosome sample the most to be checked；
If containing 1200bp or 770bp polymorphic bands in amplified production, containing Aegilops comosa 7M chromosome in chromosome sample the most to be checked.
5. according to the method described in claim 3 or 4, it is characterised in that
Amplification reaction system is 15 μ L, including the sample DNA 1 μ L of 25 ng/ μ L, and 5
U/μL Taq DNA polymerase 0.15 μ L, the dNTPs of 200 μMs, containing Mg2+10 × PCR buffer 1.5 μ L, each 1 μ L of upstream and downstream primer of 10 μMs, supplement reaction system to 15 μ L with aseptic DDW.
Method the most according to claim 5, it is characterised in that the mol ratio of the specific molecular marker of 2M, 3M, 6M and 7M chromosome is 1:1:1:1.
7. according to the method according to any one of claim 3-5, it is characterised in that: amplification program is: 94 DEG C of denaturation 3 min, subsequently 35 circulations: 94 DEG C of degeneration 45 S, 57 DEG C of annealing 45 S, 72 DEG C extend 2 min, and last 72 DEG C extend 10 min, 4 DEG C of preservations.
8. the specific molecular marker described in a claim 1 or the test kit described in claim 2 application in wheat breeding.
Application the most according to claim 8, it is characterised in that whether the specific molecular marker described in claim 1 or the test kit described in claim 2 contain the application in Aegilops comosa 2M, 3M, 6M and 7M chromosome in detection or auxiliary detection Semen Tritici aestivi.
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| CN116970733A (en) * | 2023-08-28 | 2023-10-31 | 四川农业大学 | Molecular marker primer for detecting aegilops tenuis 7M chromosome and application thereof |
| CN116949209B (en) * | 2023-08-28 | 2024-02-09 | 四川农业大学 | Molecular marker primer for detecting aegilops on top-miscanthus 5M chromosome and application thereof |
| CN116926232B (en) * | 2023-08-28 | 2024-02-09 | 四川农业大学 | Molecular marker primer for detecting aegilops on top-miscanthus 1M chromosome and application thereof |
| CN116970733B (en) * | 2023-08-28 | 2024-03-08 | 四川农业大学 | Molecular marker primer for detecting aegilops tenuis 7M chromosome and application thereof |
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