CN105695589B - Detect wheat whether reagent set and molecular labeling containing haynaldia villosa 6V#4S chromosome arm - Google Patents
Detect wheat whether reagent set and molecular labeling containing haynaldia villosa 6V#4S chromosome arm Download PDFInfo
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Abstract
The invention discloses detection wheat whether reagent set and molecular labeling containing haynaldia villosa 6V#4S chromosome arm.Whether the reagent set containing haynaldia villosa 6V#4S chromosome arm is made of detection wheat disclosed by the invention F-P and reagent a, and reagent a is G-P and/or H-P；F-P two single stranded DNAs shown in sequence 11 in sequence table and sequence 12 form；G-P two single stranded DNAs shown in sequence 13 in sequence table and sequence 14 form；H-P two single stranded DNAs shown in sequence 15 in sequence table and sequence 16 form.It is demonstrated experimentally that screening, identification and assist-breeding that reagent set of the present invention and method can be used for wheat hybridizing group have the new strain of wheat/kind of haynaldia villosa merit (such as mildew-resistance).
Description
Technical field
The present invention relates to detect in wheat genetic background whether contain haynaldia villosa 6V#4S chromosome arm in field of biotechnology
Reagent set and molecular labeling.
Background technique
Haynaldia villosa Dasypyrum villosum (L.) P.Candargy (syn.Haynaldia villosa Schur) is
One diploid kind of Tribe Triticeae Dasypyrum, 2n=2x=14.It originates from the northeast in Mediterranean, from the France south in southern Europe
Portion to the Caspian Sea, South-West Asia, Russia and Caucasus region (De Pace etc. 1995；It Frederiksen1991), is a kind of weeds
Class plant is grown in severe, arid environment (Agnieszka Gradzielewska, 2006).Due on its glume ridge and
There is a hair grown thickly on lemma top, it is therefore named it.
Haynaldia villosa contains there are many biotic and abiotic stress resistance gene and Fineness gene, is the excellent of improvement wheat
GENE SOURCES.Haynaldia villosa genome is successively transferred to the genetic background of wheat by Sears, Lukaszewski and the big equalization of Liu, is bred as
3 sets of chromosome addition systems.According to the difference in haynaldia villosa source, the wheat-haynaldia villosa that Qi etc. (1998) first cultivates Sears is attached
Adding is to be indicated with DA1V#1-DA7V#1, and the wheat-haynaldia villosa that Agricultural University Of Nanjing is cultivated is additional to use DA1V#2-DA7V#2 table
Show, Liu et al. (2011) then by A.J.Lukaszewski cultivate addition line indicated with DA1V#3-DA7V#3, applicant will before
6VS chromosome derived from Soviet Union haynaldia villosa No.1026 is named as 6V#4S (Lin etc., 2013).
The difference of 6V#1S, 6V#2S, 6V#3S and 6V#4S chromosome arm is only that source place difference.6V#1S, 6V#2S,
6V#3S with 6V#4S the short arm of a chromosome source difference also shows difference to wheat powdery mildew resistance.Carry 6V#1S and 6V#3S dyeing
The wheat of body not mildew-resistance, and the wheat with 6V#2S and 6V#4S chromosome powdery mildew show be immunized (Liu et al.,
2011)。
Chen Peidu etc. (1995) is hybridized using 6V#2 (6A) alien substitution with raising wheat No. 5, in conjunction with the gamma-radiation of hybrid generation
Processing, successfully selects mildew-resistance T6V#2S6AL translocation line, disease-resistant gene is named as Pm21 (Qi etc., 1996).Old filial piety
Using the haynaldia villosa No.1026 from the former Soviet Union cultivated durum-h. villosa Amphidiploid TH1 Deng (1996), TH2 and
6V#4 (6D) substitution line 94G22-1,94G25-1,94G32-1 and 94G33-1 of TH3 and mildew-resistance, utilize TH3 and wheat
As if the hybridization of kind 7107, backcrossing, the methods of Immature embryo culture and/Anther Culture cultivate 6V#4S6DL chromosome translocation
It is Pm97033, Pm97034 and Pm97035 (Li et al., 2005).
Although 6V#2S6AL translocation line and 6V#4S6DL translocation line are to wheat streak mosaic poison and its medium carrier-
The sensibility that wheat crimps mite is different；On Chromosome level, 2 exogenous chromosome arms are also established from different chromosomes of wheat
Linkage relationship, but the two shows all microspecies of Powdery Mildew immune, therefore is difficult to mutually to distinguish in powder mildew resistance phenotype.
The molecular labeling of based on PCR amplification is a kind of easy means for identifying exogenous chromosome.So far, for 6V#
The specific PCR mark of 2S6AL translocation line exploitation has following 7.Qi etc. is marked by the RAPD that random primer amplification is screened
OPH17；Liu et al. will be converted into stable SCAR mark SCAR after the amplified fragments recycling sequencing of the label1700；Cao etc.
(2006) it is developed based on protein serine/threonine gene (Contig17515) sequence by powdery mildew inducing expression
One can expand the codominant marker NAU/xibao15F and NAU/xibao15R of 6VS/6AS/6BS/6DS simultaneously；Chen
(2006) etc. a specifically expressed rich leucine structural domain in the disease-resistant system for carrying Pm21 is obtained using Subtractive hybridization
Gene Ta-LRR2 is translated into PCR label, can specific amplified 6VS and 6AS；Wang Chunmeis etc. (2006) are to 11 disease-resistant genes
Homologous sequence (RGA) and 17 pairs of STS primers carry out polymorphism analysis, obtain 2 stable specific molecular markers
CINAU17-1086And CINAU18-723, can specific amplified 6VS chromosome arm.The mark of the exploitations such as above-mentioned Cao, Chen and Wang Chunmei
Note separates in polyacrylamide gel.
To 6V#4S6DL translocation line, Li Hui etc. (2005) has screened the special RAPD molecular labeling of 5 6VS chromosomes,
Wherein OPAL03750It is only capable of expanding from the haynaldia villosa and wheat for carrying 6V#4S chromosome arm, becomes and be different from 6V#2S6AL
Molecular labeling.Applicant also develops 1 molecular labeling (Zhang Yunlong that can specifically distinguish 6V#2S6AL and 6V#4S6DL
Deng 2012).
Recently, Bie etc. (2015) filters out the molecular labeling that can identify 6V#2S/6V#4S/6AS/6DS simultaneously
MBH1。
Wheat powdery mildew is by biotroph powdery mildew wheat specialized form (Blumeria graminis forma
Specialis tritici) caused by a kind of worldwide fungal disease, often result in the heavy losses of wheat yield.China at present
The wheat breed that majority is widely applied is poor to the resistance of powdery mildew, limits the range and the time limit of its popularization and application.Therefore,
The powder mildew resistance of excellent wheat breed is promoted in the wheat breed of the high mildew-resistance of breeding and improvement at present, is prevention and treatment wheat white powder
Disease realizes the most safe and effective measure of wheat safety in production.So far, in wheat farm variety and wild relatives
Multiple powdery mildew resistance genes have been excavated, and have developed the molecular labeling of some disease-resistant genes.But the biological strain of powdery mildew
Variation is fast, and many disease-resistant genes are in production using shortly being overcome by new microspecies.Mildew-resistance is the one of wheat breeding
A long-term and important content.6V#2S6AL and 6V#4S6DL transposition from haynaldia villosa (Haynaldia villosa)
System shows a kind of resistance of wide spectrum, has been widely used for breeding at present because all biological strains of wheat powdery mildew are immunized
Plan.2 translocation lines are contained in the pedigree of some kinds or strain, the ownership in the anti-source of offspring has to be identified.Another aspect, due to
6V#2S with 6V#4S chromosome belongs to identical homologous group, whether resistance identical be always outstanding and the problem of do not solve, obtain special
The genetic marker of different loci helps to carry out in-depth study to this on heterochromosome arm.Therefore, no matter for breeding for disease resistance
Assisted Selection, or for theoretical research, all there is an urgent need to develop the molecular labelings for being largely specific to target chromosome.
Summary of the invention
Whether the technical problem to be solved by the present invention is to how to detect in wheat genetic background containing haynaldia villosa 6V#4S dye
Colour solid arm.
In order to solve the above technical problems, present invention firstly provides detections or auxiliary detection wheat whether to contain haynaldia villosa
The reagent set of 6V#4S chromosome arm, entitled reagent set 2.The reagent set 2 is made of F-P and reagent a, described
Reagent a is G-P and/or H-P；
The F-P two single stranded DNAs shown in sequence 11 in sequence table and sequence 12 form；
The G-P two single stranded DNAs shown in sequence 13 in sequence table and sequence 14 form；
The H-P two single stranded DNAs shown in sequence 15 in sequence table and sequence 16 form.
Each single stranded DNA in the reagent set 2 can independent packaging, can also be packaged together；It can also will be therein each
Primer pair is individually packed.The molal quantity ratio of each single stranded DNA can be carried out according to actually detected sample in the reagent set 2
It adjusts, the molal quantity of each single stranded DNA also can be identical in the reagent set 2, mole of each primer pair in the reagent set 2
Number also can be identical.
In order to solve the above technical problems, the present invention also provides detections or auxiliary detection wheat whether to contain haynaldia villosa 6V#
The primer pair of 4S chromosome arm, the primer pair are the F-P.
In order to solve the above technical problems, the present invention also provides detections or auxiliary detection wheat whether to contain haynaldia villosa 6V#
The reagent set of 4S chromosome arm, entitled reagent set 2-1.The reagent set 2-1 is by the reagent set 2 or described
F-P and Y1 is formed；The Y1 is at least one of B-P, C-P, D-P and E-P；
The B-P two single stranded DNAs shown in sequence 3 in sequence table and sequence 4 form；
The C-P two single stranded DNAs shown in sequence 5 in sequence table and sequence 6 form；
The D-P two single stranded DNAs shown in sequence 7 in sequence table and sequence 8 form；
The E-P two single stranded DNAs shown in sequence 9 in sequence table and sequence 10 form.
Each single stranded DNA in the reagent set 2-1 can independent packaging, can also be packaged together；It can also will be therein
Each primer pair is individually packed.The molal quantity ratio of each single stranded DNA can be according to actually detected sample in the reagent set 2-1
It is adjusted, the molal quantity of each single stranded DNA also can be identical in the reagent set 2-1, each primer in the reagent set 2-1
Pair molal quantity also can be identical.
In order to solve the above technical problems, the present invention also provides detections or auxiliary detection wheat whether to contain haynaldia villosa 6V#
The system of 4S chromosome arm.The system is system B1, system B2 or system B3；
The system B1 is made of the reagent set 2 and reagent needed for progress PCR amplification and/or instrument；
The system B2 is made of the F-P and reagent needed for progress PCR amplification and/or instrument；
The system B3 is made of the reagent set 2-1 and reagent needed for progress PCR amplification and/or instrument.
In above system, reagent needed for the carry out PCR amplification in the system B1, the system B2 and the system B3
It can be archaeal dna polymerase or the reagent (such as 2x Taq MasterMix) containing archaeal dna polymerase.2x Taq MasterMix can be
Beijing CoWin Bioscience Co., Ltd.'s product, article No. CW0682A.The system B1, the system B2 and the system
Instrument needed for carry out PCR amplification in system B3 can be PCR instrument.The PCR instrument can be Bio-RAD T100TM Thermal
Cycler。
In order to solve the above technical problems, the present invention also provides haynaldia villosa molecular labelings.The haynaldia villosa molecular labeling is
For molecular labeling b1, molecular labeling b2 or molecular labeling b3；
The molecular labeling b1 is made of F and b, and the b is that G and H is formed；
The F is DNA molecular shown in sequence 22, which is by template of haynaldia villosa genomic DNA with the F-
P carries out the DNA molecular that PCR amplification obtains；
The G is DNA molecular shown in sequence 23, which is by template of haynaldia villosa genomic DNA with the G-
P carries out the DNA molecular that PCR amplification obtains；
The H is DNA molecular shown in sequence 24, which is by template of haynaldia villosa genomic DNA with the H-
P carries out the DNA molecular that PCR amplification obtains；
The molecular labeling b2 is the F；
The molecular labeling b3 is made of the molecular labeling b1 or the molecular labeling b2 and V1；The V1 is B, C, D
At least one of with E；
The B is DNA molecular shown in sequence 18, which is by template of haynaldia villosa genomic DNA with the B-
P carries out the DNA molecular that PCR amplification obtains；
The C is DNA molecular shown in sequence 19, which is by template of haynaldia villosa genomic DNA with the C-
P carries out the DNA molecular that PCR amplification obtains；
The D is DNA molecular shown in sequence 20, which is by template of haynaldia villosa genomic DNA with the D-
P carries out the DNA molecular that PCR amplification obtains；
The E is DNA molecular shown in sequence 21, which is by template of haynaldia villosa genomic DNA with the E-
P carries out the DNA molecular that PCR amplification obtains.
In order to solve the above technical problems, the present invention also provides detections or auxiliary detection wheat whether to contain haynaldia villosa 6V#
The method of 4S chromosome arm.The method is following T1) or T2):
T1) include following T11) and T12):
T11) respectively using the genomic DNA of wheat to be measured, reference wheat and haynaldia villosa as template, distinguished with 3 kinds of primer pairs
PCR amplification is carried out, the wheat PCR product to be measured of 3 kinds of primer pairs, the reference wheat PCR product of 3 kinds of primer pairs are obtained
With the haynaldia villosa PCR product of 3 kinds of primer pairs；The reference wheat is the wheat without haynaldia villosa 6V#4S chromosome arm, is made
For the reference for determining haynaldia villosa specific band；
T12) by the wheat PCR product to be measured of 3 kinds of primer pairs, the reference wheat PCR product of 3 kinds of primer pairs and
The haynaldia villosa PCR product of 3 kinds of primer pairs carries out electrophoresis, and the wheat to be measured of 3 kinds of primer pairs is determined according to electrophoresis result
In PCR product whether the specific band containing the corresponding primer pair of haynaldia villosa, if the wheat PCR to be measured of 3 kinds of primer pairs is produced
Specific band containing the corresponding primer pair of haynaldia villosa in the wheat PCR product to be measured of at least a kind of primer pair in object, it is described to
Survey wheat contains or candidate contains haynaldia villosa 6V#4S chromosome arm；If in the wheat PCR product to be measured of 3 kinds of primer pairs
The specific band of the corresponding primer pair of haynaldia villosa is not contained, and the wheat to be measured is free of or the candidate haynaldia villosa 6V#4S that is free of is dyed
Body arm；
3 kinds of primer pairs are the F-P, the G-P and the H-P；
The specific band of the corresponding primer pair of the haynaldia villosa is that the haynaldia villosa PCR product of same primer pair contains but described
The band that the PCR product of the reference wheat of same primer pair does not contain；The special item of the corresponding primer pair of the haynaldia villosa
The band that the specially described F-P of band, the G-P or the H-P are shown in electrophoresis；
T2) include following T21) and T22):
T21) using the genomic DNA of wheat to be measured as template, PCR amplification is carried out respectively with 3 kinds of primer pairs, obtains institute
State the wheat PCR product to be measured of 3 kinds of primer pairs；
T22) detect T21) 3 kinds of primer pairs wheat PCR product to be measured sequence, such as 3 kinds of primer pairs
Containing at least one of the F, the G and described H in wheat PCR product to be measured, the wheat to be measured contains or candidate contains
There is haynaldia villosa 6V#4S chromosome arm；As 3 kinds of primer pairs wheat PCR product to be measured in be free of the F, the G and institute
It states any one of H or appoints several, the wheat to be measured is free of or the candidate 6V#4S chromosome arm for being free of haynaldia villosa.
In the above method, the primer annealing temperature that the PCR amplification uses can be 60-64 DEG C.
In the above method, the annealing temperature that PCR amplification is carried out with the F-P and the G-P can be 60 DEG C, with the H-
The annealing temperature that P carries out PCR amplification can be 64 DEG C.
In the above method, the reaction system of the PCR amplification can 2x Taq MasterMix, genomic DNA, primer
To and water composition.The concentration of the forward primer of each primer pair can be 0.4 μM in the reaction system, the reverse primer of each primer pair
Concentration can be 0.4 μM, the 2x Taq MasterMix concentration can for 2 ×, the concentration of genomic DNA can be 8ng/ μ l.Institute
The PCR reaction condition for stating PCR amplification can are as follows: 95 DEG C of 5min；94 DEG C of 30s, 58-60 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃
5min。
In order to solve the above technical problems, the present invention also provides the reagent sets 2, the reagent set 2-1, the F-
P, whether the system, the haynaldia villosa molecular labeling or the detection or auxiliary detection wheat contain haynaldia villosa 6V#4S dyeing
Following Z1 of the method for body arm or the application of Z2:
Z1, detecting or assisting whether detection wheat contains the application in haynaldia villosa 6V#4S chromosome arm；
Z2, the application in wheat breeding.
In order to solve the above technical problems, the present invention also provides the breeding methods of wheat.The breeding method packet of the wheat
Include with according to detection or auxiliary detection wheat whether containing of obtaining of the method containing haynaldia villosa 6V#4S chromosome arm or candidate contains
There is the wheat to be measured of haynaldia villosa 6V#4S chromosome arm to carry out breeding as parent.
In the present invention, the wheat to be measured and the wheat can be haynaldia villosa or be improved using haynaldia villosa
Wheat, as 6V#2S6AL translocation line, 6V#4S6DL translocation line Pm97033, Yang Mai 22, interior wheat 836, Yang Mai 18, CB037 or
Golden standing grain 9123.
Present invention obtains the recruit of 8 kinds of haynaldia villosa 6VS chromosome arms labels: A, B, C, D, E, F, G and H.The present invention
Detect in wheat to be measured whether contain haynaldia villosa 6V#4S using the detection or auxiliary of above-mentioned molecular labeling F, G and H and its primer pair
The method of chromosome arm detects whether wheat to be measured contains haynaldia villosa 6V#4S chromosome with the label MBH1 using identification 6V#4S
The result of the method for arm is completely the same.Reagent set and method of the invention can be used for wheat hybridizing group screening, identification with
Assist-breeding has the new strain of wheat/kind of haynaldia villosa merit (such as mildew-resistance).
Detailed description of the invention
Fig. 1 be reagent set 1 in A-P, B-P and C-P detect wheat whether the electrophoresis result containing H. villosa chromosome arm.
Fig. 2 be in reagent set 1 D-P and E-P detect wheat whether the electrophoresis result containing H. villosa chromosome arm.
In Fig. 1 and Fig. 2, swimming lane M is DNA molecular amount standard (DM2000bp), as if swimming lane 1 is 7107, swimming lane 2 is
Haynaldia villosa NO.1026 (HV-S), swimming lane 3 are 6V#4S6DL translocation line Pm97033, and swimming lane 4 is to raise wheat 22, and swimming lane 5 is equal
For 6V#2S6AL translocation line, swimming lane 6 is interior wheat 836, and swimming lane 7 is to raise wheat 18, and swimming lane 8 is CB037, and swimming lane 9 is
Golden standing grain 9123.
As if Fig. 3 is wheat 7107, the powdery-mildew-resistance wheat translocation line Pm97033, cluster for feeling powdery mildew using 1 couple of reagent set
The part electrophoretogram that dirty wheat NO.1026 and 6V#2S6AL translocation line is detected.Wherein, as if swimming lane 1 indicates 7107, swimming lane 2
Indicate that Pm97033, swimming lane 3 indicate that haynaldia villosa NO.1026, swimming lane 4 indicate that 6V#2S6AL translocation line, swimming lane M indicate DNA molecular
Amount standard.
Fig. 4 is to the peaceful spring Improved lines in peaceful spring number and its mildew-resistance using A-P in reagent set 1 (to feel powdery mildew
Peaceful spring wheat be the BC that hybridizes of wheat CB037 for orienting backcross parent with carrying 6V#2S6AL translocation chromosome5F6Generation)
The testing result of middle H. villosa chromosome arm.Wherein, swimming lane M is DNA molecular amount standard DM (2000bp), and swimming lane 1-5 is peaceful
No. 4 Improved lines of spring, swimming lane 6-10 are No. 47 Improved lines of Ning Chun, and swimming lane 11-15 is 50 Improved lines of Ning Chun, and swimming lane 16 is Ning Chun
No. 4, swimming lane 17 is the peaceful spring 47, and swimming lane 18 is the peaceful spring 50, and swimming lane 19 is CB037, and swimming lane 20 is haynaldia villosa, and swimming lane 21 is
Pm97033。
Fig. 5 be reagent set 2 in F-P, G-P and H-P detect wheat whether the electrophoresis result containing H. villosa chromosome arm.
Wherein, swimming lane M is DNA molecular amount standard (DM2000bp), as if swimming lane 1 is 7107, swimming lane 2 is haynaldia villosa NO.1026 (HV-S),
Swimming lane 3 is 6V#4S6DL translocation line Pm97033, and swimming lane 4 is to raise wheat 22, and swimming lane 5 is 6V#2S6AL translocation line, and swimming lane 6 is
Interior wheat 836, swimming lane 7 are to raise wheat 18, and swimming lane 8 is CB037, and swimming lane 9 is golden standing grain 9123.
As if Fig. 6 is to utilize 2 pair 7107 of reagent set, anti-disease wheat translocation line Pm97033, haynaldia villosa NO.1026 and 6V#
The electrophoretogram that 2S6AL translocation line is detected.Wherein, as if swimming lane 1 indicates 7107, swimming lane 2 indicates anti-disease wheat translocation line
Pm97033, swimming lane 3 indicate that haynaldia villosa NO.1026, swimming lane 4 indicate that 6V#2S6AL translocation line, swimming lane M indicate DNA molecular amount mark
It is quasi-.
Fig. 7 is the testing result using MBH1 label to wheat Haynaldia villosa chromosome arm.Wherein, swimming lane M is DNA molecular amount
Standard (DM2000bp), as if swimming lane 1 is 7107, swimming lane 2 is haynaldia villosa NO.1026 (HV-S), and swimming lane 3 is 6V#4S6DL transposition
It is Pm97033, swimming lane 4 is to raise wheat 22, and swimming lane 5 is 6V#2S6AL translocation line, and swimming lane 6 is interior wheat 836, and swimming lane 7 is to raise wheat 18,
Swimming lane 8 is CB037, and swimming lane 9 is golden standing grain 9123.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
2x Taq MasterMix in following embodiments is Beijing CoWin Bioscience Co., Ltd.'s product, article No.
For CW0682A.
Embodiment 1, wheat whether the detection containing H. villosa chromosome arm
One, detection wheat whether the preparation of the reagent set containing H. villosa chromosome arm
Detecting wheat, whether the reagent set containing H. villosa chromosome arm is reagent set 1 and reagent set 2, complete examination
Agent 1 is made of A-P, B-P, C-P, D-P and E-P；
A-P two single stranded DNAs shown in sequence 1 in sequence table and sequence 2 form；
B-P two single stranded DNAs shown in sequence 3 in sequence table and sequence 4 form；
C-P two single stranded DNAs shown in sequence 5 in sequence table and sequence 6 form；
D-P two single stranded DNAs shown in sequence 7 in sequence table and sequence 8 form；
E-P two single stranded DNAs shown in sequence 9 in sequence table and sequence 10 form；
Reagent set 2 is made of F-P, G-P and H-P；
F-P two single stranded DNAs shown in sequence 11 in sequence table and sequence 12 form；
G-P two single stranded DNAs shown in sequence 13 in sequence table and sequence 14 form；
H-P two single stranded DNAs shown in sequence 15 in sequence table and sequence 16 form.
The equal independent packaging of each single stranded DNA in reagent set 1 and reagent set 2, each single stranded DNA rubs in each primer pair
Your ratio is 1:1.When carrying out PCR amplification as template using haynaldia villosa genomic DNA,
DNA molecular shown in sequence 17 can be amplified using A-P；
DNA molecular shown in sequence 18 can be amplified using B-P；
DNA molecular shown in sequence 19 can be amplified using C-P；
DNA molecular shown in sequence 20 can be amplified using D-P；
DNA molecular shown in sequence 21 can be amplified using E-P；
DNA molecular shown in sequence 22 can be amplified using F-P；
DNA molecular shown in sequence 23 can be amplified using G-P；
DNA molecular shown in sequence 24 can be amplified using H-P.
Two, wheat whether the detection containing haynaldia villosa 6VS chromosome arm
1, the selection of sample is detected
Wheat to be measured: 6V#2S6AL translocation line is also referred to as southern agriculture translocation line (NY is cultivated by Agricultural University Of Nanjing)
(Chen PD,Qi LL,Zhou B,Zhang SZ,Liu DJ(1995)Development and molecular
cytogenetic Analysis of wheat-Haynaldia villosa 6VS/6AL.Translocation lines
specifying resistance to powdery mildew.Theor Appl Genet 91:1125–1128)
Carry 6V#4S6DL chromosome anti-disease wheat translocation line Pm97033 (6V#4S6DL translocation line, hereinafter referred to as
Pm97033 is the translocation line cultivated using the haynaldia villosa that the former Soviet Union introduces, Li H, Chen X, Xin ZY, Ma YZ, Xu
HJ,Chen XY,Jia X(2005)Development and identification of wheat–Haynaldia
villosa T6DL·6VS chromosome translocation lines conferring resistance to
powdery mildew.Plant Breed 124:203–205)
Wheat 22:Pm97033 offspring is raised, state examines wheat 2012004, LX-river area agricultural sciences institute, Jiangsu Province, and powdery mildew is exempted from
Epidemic disease.
Golden standing grain 9123: (state examines wheat 2012008 to stone 4185/92R137//stone 4185, contains Pm21, Hebei province's agricultural and forest science
Genetics and physiology research institute, institute, 92R137 is 6V#2S6AL translocation line in pedigree), powdery mildew is immune.
Interior wheat 836: state examines wheat 2008001, Neijiang City in Sichuan Province Academy of Agricultural Sciences
Raise wheat 18: Anhui wheat 2008001, LX-river area agricultural sciences institute, Jiangsu Province
Middle wheat 113 (wheat 2013001 is examined in capital), crop science research institute, the Chinese Academy of Agricultural Sciences
CB037 (6V#2S6AL translocation line) (Zhang Yunlong, Wang Meijiao, Zhang Yue, Chu Cuiping, Lin Zhishan (communication author), Xu
Fine jade virtue, Ye Xingguo, old filial piety, constitution save.Different haynaldia villosa 6VS chromosome arms powder mildew resistance exceptional function label exploitation and
Using, Acta Agronomica Sinica, 2012,38 (10): 1827-1832.)
Reference wheat: as if susceptible wheat parent 7107 (as if hereinafter referred to as 7107, common wheat) (old filial piety, Shi Ainong, still
The vertical people, Resistant reaction of the haynaldia villosa to different Powdery Mildew fungus strains and its expression under wheat genetic background, Plant Pathology
Report, 1997.27 (1): 17~22).
Haynaldia villosa: haynaldia villosa NO.1026 (HV-S) (old filial piety, Xu Huijun, Du Lipu, Shang Limin, Han Bin, Shi Ainong, Xiao's generation
With, using tissue culture technique to common wheat import haynaldia villosa mildew-resistance gene research, Scientia Agricultura Sinica, 1996
(5), 29:1-8.)
2, detect whether wheat contains haynaldia villosa 6VS chromosome arm using reagent set 1
Whether 2.1 detection wheats contain H. villosa chromosome arm
Extraction step 1 wheat to be measured (6V#4S6DL translocation line Pm97033, Yang Mai 22,6V#2S6AL translocation line,
Interior wheat 836, Yang Mai 18, middle wheat 113, CB037 and Jin He 9123), the genomic DNA of reference wheat and haynaldia villosa, utilize step
One reagent set 1 carries out PCR amplification, and PCR reaction system is 20 μ l reaction systems, specifically: including ddH2O 14μl,2x
10 μ l of Taq MasterMix, forward primer (10 μm of ol × L-1) 0.4 μ l, reverse primer (10 μm of ol × L-1) 0.4 μ l, gene
Group DNA (100ng/ μ l) 1 μ l.
PCR reaction condition are as follows: 95 DEG C of denaturation 5min；94 DEG C of denaturation 30s, the corresponding annealing temperature of primer pair are annealed 30s, and 72 DEG C
Extend 1min, 35 circulations；Last 72 DEG C of extensions 5min.The PCR instrument for carrying out PCR amplification is Bio-RAD T100TM Thermal
After Cycler, PCE are expanded, the PCR product of each wheat of each primer pair is existed using the plain agar sugar gel of 1-3%
Under 120V, electrophoresis 30min in 1 × TAE buffer.The electrophoresis result of part wheat is as depicted in figs. 1 and 2.
The annealing temperature of the reagent set 1 of step 1 is as shown in table 1.
Table 1, step 1 reagent set 1 annealing temperature
| Primer pair title | Sequence | Annealing temperature |
| A-P | Forward primer A-F, sequence 1；Reverse primer A-R, sequence 2 | 60℃ |
| B-P | Forward primer B-F, sequence 3；Reverse primer B-R, sequence 4 | 58℃ |
| C-P | Forward primer C-F, sequence 5；Reverse primer C-R, sequence 6 | 58℃ |
| D-P | Forward primer D-F, sequence 7；Reverse primer D-R, sequence 8 | 58℃ |
| E-P | Forward primer E-F, sequence 9；Reverse primer E-R, sequence 10 | 58℃ |
The haynaldia villosa specific band of various primer pairs, the haynaldia villosa specific band of primer pair A-P are determined according to electrophoresis result
To contain in the A-P PCR product of haynaldia villosa and band that the A-P PCR product of reference wheat is free of, which is recycled and is carried out
Sequencing, the band are DNA molecular shown in sequence 17 (band is named as haynaldia villosa specific band A)；The cluster of primer pair B-P
Dirty wheat specific band be haynaldia villosa B-P PCR product in contain and band that the B-P PCR product of reference wheat is free of, by this
Band recycling is sequenced, which is DNA molecular shown in sequence 18 (band is named as haynaldia villosa specific band B)；
The haynaldia villosa specific band of primer pair C-P be haynaldia villosa C-P PCR product in contain and the C-P PCR product of reference wheat not
Band recycling is sequenced the band contained, which is that the band (is named as tuft by DNA molecular shown in sequence 19
Wheat specific band C)；The haynaldia villosa specific band of primer pair D-P be haynaldia villosa D-P PCR product in contain and reference wheat
Band recycling is sequenced the band that D-P PCR product is free of, which is DNA molecular shown in sequence 20 (by this
Band is named as haynaldia villosa specific band D)；The haynaldia villosa specific band of primer pair E-P be haynaldia villosa E-P PCR product in contain
And the band that the E-P PCR product of reference wheat is free of, band recycling is sequenced, which is shown in sequence 21
The band (is named as haynaldia villosa specific band E) by DNA molecular.
According to the above results, the haynaldia villosa for whether containing each primer pair in each primer pair PCR product of each wheat to be measured determined
Specific band, the results are shown in Table 2.
Whether table 2, each wheat contain each PCR primer to the result of haynaldia villosa specific band
In table 2, A indicates to contain haynaldia villosa specific band A, and B indicates to contain haynaldia villosa specific band B, and C expression contains tuft
Wheat specific band C, D indicate to indicate that F expression contains haynaldia villosa containing haynaldia villosa specific band E containing haynaldia villosa specific band D, E
Specific band F, G expression contains haynaldia villosa specific band G；It indicates not containing haynaldia villosa specific band.
The results show that in wheat to be measured, 6V#4S6DL translocation line Pm97033, Yang Mai 22,6V#2S6AL transposition
System, interior wheat 836, Yang Mai 18, CB037 and Jin He 9123 contain haynaldia villosa specific band A, B, C, D and E, and middle wheat 113 is free of
There are any one of haynaldia villosa specific band A, B, C, D and E.
2.2 target fragments are haynaldia villosa 6VS chromosome arm
With each primer pair of reagent set 1, with haynaldia villosa NO.1026,6V#2S6AL translocation line, (NY is a benefit respectively
The translocation line cultivated with the haynaldia villosa from Britain Camb botanical garden, Chen PD, Qi LL, Zhou B, Zhang SZ, Liu DJ
(1995)Development and molecular cytogenetic Analysis of wheat-Haynaldia
villosa 6VS/6AL.Translocation lines specifying resistance to powdery
91:1125-1128 mildew.Theor Appl Genet, exogenous chromosome fragment are only 6V#2S chromosome arm), carry
(hereinafter referred to as Pm97033 is one and is introduced using the former Soviet Union anti-disease wheat translocation line Pm97033 of 6V#4S6DL chromosome
Haynaldia villosa cultivate translocation line, Li H, Chen X, Xin ZY, Ma YZ, Xu HJ, Chen XY, Jia X (2005)
Development and identification of wheat–Haynaldia villosa T6DL·6VS
chromosome translocation lines conferring resistance to powdery mildew.Plant
124:203-205 Breed, exogenous chromosome fragment are only 6V#4S chromosome arm) as and if 7107 (old filial piety, Shi Ainong, Shang Li
The people, Resistant reaction of the haynaldia villosa to different Powdery Mildew fungus strains and its expression under wheat genetic background, Plant Pathology,
1997.27 (1): 17~22, without from haynaldia villosa chromosome segment) genomic DNA be template according to step 2.1
Reaction system and reaction condition carry out PCR amplification, the electrophoresis result of A-P, C-P and D-P is as shown in Figure 3.
Sequencing result show using A-P only haynaldia villosa NO.1026, NY and carry 6V#4S6DL chromosome it is disease-resistant
Expanded in the PCR reaction of Wheat Translocation Line Pm97033 band (band shown in arrow in A-P in Fig. 3, as if 7107
PCR reaction in without the band) sequence be sequence 17, show DNA molecular shown in sequence 17 (i.e. haynaldia villosa specific band A)
For haynaldia villosa 6VS chromosome arm；Only in haynaldia villosa NO.1026, NY and the disease-resistant small of 6V#4S6DL chromosome is carried using B-P
Wheat translocation line Pm97033 PCR reaction in expand band (as if 7107 PCR reaction in without the band) sequence be
Sequence 18 shows that DNA molecular shown in sequence 18 (i.e. haynaldia villosa specific band B) is haynaldia villosa 6VS chromosome arm；Utilize C-P
Only in the PCR reaction of haynaldia villosa NO.1026, NY and the anti-disease wheat translocation line Pm97033 for carrying 6V#4S6DL chromosome
The sequence for expanding obtained band (band shown in arrow in C-P in Fig. 3, as if without the band in 7107 PCR reaction) is sequence
Column 19 show that DNA molecular shown in sequence 19 (i.e. haynaldia villosa specific band C) is haynaldia villosa 6VS chromosome arm；Only using D-P
Expand in the PCR reaction of haynaldia villosa NO.1026, NY and the anti-disease wheat translocation line Pm97033 for carrying 6V#4S6DL chromosome
The sequence for increasing obtained band (band shown in arrow in D-P in Fig. 3, as if without the band in 7107 PCR reaction) is sequence
20, show that DNA molecular shown in sequence 20 (i.e. haynaldia villosa specific band D) is haynaldia villosa 6VS chromosome arm；Only existed using E-P
It is expanded in the PCR reaction of the anti-disease wheat translocation line Pm97033 of haynaldia villosa NO.1026, NY and carrying 6V#4S6DL chromosome
The sequence of obtained band (as if without the band in 7107 PCR reaction) is sequence 21, shows DNA shown in sequence 21 points
Sub (i.e. haynaldia villosa specific band E) is haynaldia villosa 6VS chromosome arm.
The detection of H. villosa chromosome arm in 2.3 peaceful spring number and its peaceful spring powder mildew resistance Improved lines
Choose 5 plants of Ningchun4 Improved lines, 5 plants of peaceful No. 47 Improved lines of spring, 5 plants of 50 Improved lines of peaceful spring, Ningchun4s, Ning Chun 47
Number and peaceful spring No. 50 detections of H. villosa chromosome arm are carried out using A-P, wherein Ningchun4 Improved lines are that utilization contains 6V#
The wheat CB037 of 2S6AL chromosome hybridizes with Ningchun4, then is returned the strain improved with Ningchun4, with
Same procedure, No. 47 Improved lines of Ning Chun are using CB037 to No. 47 strains improved of peaceful spring, and 50 Improved lines of Ning Chun are
Using CB037 to No. 50 strains improved of peaceful spring.With CB037, haynaldia villosa NO.1026 and Pm97033 (6V#4S
6DL translocation line) as control, detection method is the same as step 2.1.
As a result (Fig. 4) is shown, in 5 plants of Ningchun4 Improved lines, 5 plants of peaceful No. 47 Improved lines of spring and 5 plants of 50 Improved lines of peaceful spring
Containing haynaldia villosa specific band A, and Ningchun4, Ning Chun 47 and peaceful spring No. 50 do not contain haynaldia villosa specific band A, show
This 5 plants of Ningchun4 Improved lines, 5 plants of peaceful No. 47 Improved lines of spring and 5 plants of 50 Improved lines of peaceful spring contain haynaldia villosa 6VS chromosome arm.
3, detect whether wheat contains haynaldia villosa 6V#4S chromosome arm using reagent set 2
Whether 3.1 detection wheats contain H. villosa chromosome arm
The genomic DNA of each wheat to be measured of extraction step 1, reference wheat and haynaldia villosa, utilizes the complete examination of step 1
Agent 2 carries out PCR amplification, and PCR reaction system is 20 μ l reaction systems, specifically: including ddH2O 14μl,2x Taq
10 μ l of MasterMix, forward primer (10 μm of ol × L-1) 0.3 μ l, reverse primer (10 μm of ol × L-1) 0.3 μ l, genomic DNA
(100ng/μl)1μl.PCR reaction condition are as follows: 95 DEG C of denaturation 5min；94 DEG C of denaturation 30s, the corresponding annealing temperature annealing of primer pair
30s, 72 DEG C of extension 1min, 35 circulations；Last 72 DEG C of extensions 5min.The PCR instrument for carrying out PCR amplification is Bio-RAD T100TM
The PCR product of each wheat of each primer pair after PCE is expanded, is utilized the plain agar sugar of 1-3% by Thermal Cycler
Gel is at 120V, electrophoresis 30min in 1 × TAE buffer.The electrophoresis result of part wheat is as shown in Figure 5.
The annealing temperature of the reagent set 2 of step 1 is as shown in table 3.
Table 3, step 1 reagent set 2 annealing temperature
| Primer pair title | Sequence | Annealing temperature |
| F-P | Forward primer F-F, sequence 11；Reverse primer F-R, sequence 12 | 60℃ |
| G-P | Forward primer G-F, sequence 13；Reverse primer G-R, sequence 14 | 60℃ |
| H-P | Forward primer H-F, sequence 15；Reverse primer H-R, sequence 16 | 64℃ |
The haynaldia villosa specific band of various primer pairs, the haynaldia villosa specific band of primer pair F-P are determined according to electrophoresis result
To contain in the F-P PCR product of haynaldia villosa and band that the F-P PCR product of reference wheat is free of, which is recycled and is carried out
Sequencing, the band are DNA molecular shown in sequence 22 (band is named as haynaldia villosa specific band F)；The cluster of primer pair G-P
Dirty wheat specific band be haynaldia villosa G-P PCR product in contain and band that the G-P PCR product of reference wheat is free of, by this
Band recycling is sequenced, which is DNA molecular shown in sequence 23 (band is named as haynaldia villosa specific band G)；
The haynaldia villosa specific band of primer pair H-P be haynaldia villosa H-P PCR product in contain and the H-P PCR product of reference wheat not
Band recycling is sequenced the band contained, which is that the band (is named as tuft by DNA molecular shown in sequence 24
Wheat specific band H).
According to the above results, the haynaldia villosa for whether containing each primer pair in each primer pair PCR product of each wheat to be measured determined
Specific band, the results are shown in Table 4.
Whether table 4, each wheat contain each PCR primer to the result of haynaldia villosa specific band
In table 4, F indicates to contain haynaldia villosa specific band F, and G indicates to contain haynaldia villosa specific band G, and H expression contains tuft
Wheat specific band H；It indicates not containing haynaldia villosa specific band.
The results show that in wheat to be measured, 6V#4S6DL translocation line Pm97033 with raise wheat 22 containing haynaldia villosa it is special
Band F, G and H, and 6V#2S6AL translocation line, interior wheat 836, Yang Mai 18, CB037, Jin He 9123 and middle wheat 113 do not contain
Any one of haynaldia villosa specific band F, G and H.
3.2 target fragments are haynaldia villosa 6V#4S chromosome arm
With each primer pair of reagent set 2, with haynaldia villosa NO.1026,6V#2S6AL translocation line, (NY is a benefit respectively
The translocation line cultivated with the haynaldia villosa from Britain Camb botanical garden, Chen PD, Qi LL, Zhou B, Zhang SZ, Liu DJ
(1995)Development and molecular cytogenetic Analysis of wheat-Haynaldia
villosa 6VS/6ALtranslocation lines specifying resistance to powdery
91:1125-1128 mildew.Theor Appl Genet, exogenous chromosome fragment are only 6V#2S chromosome arm), carry
(hereinafter referred to as Pm97033 is one and is introduced using the former Soviet Union anti-disease wheat translocation line Pm97033 of 6V#4S6DL chromosome
Haynaldia villosa cultivate translocation line, Li H, Chen X, Xin ZY, Ma YZ, Xu HJ, Chen XY, Jia X (2005)
Development and identification of wheat–Haynaldia villosa T6DL·6VS
chromosome translocation lines conferring resistance to powdery mildew.Plant
124:203-205 Breed, exogenous chromosome fragment are only 6V#4S chromosome arm) as and if susceptible wheat parent 7107 it is (old
Filial piety, Shi Ainong, Shang Limin, Resistant reaction of the haynaldia villosa to different Powdery Mildew fungus strains and its table under wheat genetic background
Reach, Plant Pathology, 1997.27 (1): 17~22, without from haynaldia villosa chromosome segment) genomic DNA
PCR amplification is carried out according to the reaction system and reaction condition of step 2.1 for template, as a result as shown in Figure 6.
Sequencing result is shown, using F-P only in the anti-disease wheat of haynaldia villosa NO.1026 and carrying 6V#4S6DL chromosome
Expanded in the PCR reaction of translocation line Pm97033 band (band shown in arrow in F-P in Fig. 6, as if 7107 and NY's
PCR reaction in without the band) sequence be sequence 22, show (the i.e. haynaldia villosa specific band of DNA molecular shown in sequence 22
It F) is haynaldia villosa 6V#4S chromosome arm；Only in haynaldia villosa NO.1026 and the disease-resistant of 6V#4S6DL chromosome is carried using G-P
Expanded in the PCR reaction of Wheat Translocation Line Pm97033 band (band shown in arrow in G-P in Fig. 6, as if in 7107 Hes
NY PCR reaction in without the band) sequence be sequence 23, show that (i.e. haynaldia villosa is special for DNA molecular shown in sequence 23
Band G) it is haynaldia villosa 6V#4S chromosome arm；Only in haynaldia villosa NO.1026 and 6V#4S6DL chromosome is carried using H-P
Expanded in the PCR reaction of anti-disease wheat translocation line Pm97033 band (band shown in arrow in H-P in Fig. 6, as if
7107 and NY PCR reaction in without the band) sequence be sequence 24, show DNA molecular (i.e. tuft shown in sequence 24
Wheat specific band 24) it is haynaldia villosa 6V#4S chromosome arm.
Show 6V#4S6DL translocation line Pm97033 and raises wheat 22 containing haynaldia villosa 6V#4S chromosome arm, and 6V#
2S6AL translocation line, interior wheat 836, Yang Mai 18, CB037, Jin He 9123 and middle wheat 113 do not contain haynaldia villosa 6V#4S chromosome
Arm.
4, the further verifying of wheat Haynaldia villosa 6VS chromosome arm to be measured and haynaldia villosa 6V#4S chromosome arm
MBH1 is label (Tongde Bie etc., the Mol Breeding of an identification 6V#2S and 6V#4S of the exploitations such as Bie
(2015) 35:189, DOI 10.1007/s11032-015-0385-3), using the label to the wheat to be measured in step 1, ginseng
It is detected than wheat and haynaldia villosa NO.1026 (HV-S), the electrophoresis detection result of part wheat is as shown in Figure 7.
The results show that as if 7107 are free of the band of 271bp and 341bp, haynaldia villosa NO.1026,6V#4S6DL translocation line
Pm97033 with raise the band containing 271bp of wheat 22 but do not contain 341bp band, 6V#2S6AL translocation line, interior wheat 836,
It raises wheat 18, CB037, Jin He 9123 band containing 341bp but does not contain the band of 271bp, middle wheat 113 did not both contain
The band of 271bp does not contain the band of 341bp yet.Show wheat 6V#4S6DL translocation line Pm97033 to be measured and raises wheat 22
Containing haynaldia villosa 6V#4S chromosome arm, and 6V#2S6AL translocation line, interior wheat 836, Yang Mai 18, CB037 and Jin He 9123 contain
Have containing haynaldia villosa 6V#2S chromosome arm, may further learn wheat 6V#4S6DL translocation line Pm97033, Yang Mai to be measured
22,6V#2S6AL translocation line, interior wheat 836, Yang Mai 18, CB037 and Jin He 9123 contain haynaldia villosa 6VS chromosome arm；In
Wheat 113 does not contain haynaldia villosa 6VS chromosome arm.It further illustrates, reagent set 1 and reagent set 2 are in detection wheat Haynaldia villosa
The feasibility of 6VS chromosome arm and reagent set 2 in detection wheat Haynaldia villosa 6V#4S chromosome arm.
Claims (16)
1. detection or auxiliary detection wheat whether the reagent set containing haynaldia villosa 6V#4S chromosome arm, by F-P and reagent a group
At the reagent a is G-P and H-P；
The F-P two single stranded DNAs shown in sequence 11 in sequence table and sequence 12 form；
The G-P two single stranded DNAs shown in sequence 13 in sequence table and sequence 14 form；
The H-P two single stranded DNAs shown in sequence 15 in sequence table and sequence 16 form.
2. detection or auxiliary detection wheat whether the primer pair containing haynaldia villosa 6V#4S chromosome arm, be F-P；
The F-P two single stranded DNAs shown in sequence 11 in sequence table and sequence 12 form.
3. detection or auxiliary detection wheat whether the reagent set containing haynaldia villosa 6V#4S chromosome arm, as described in claim 1
F-P described in reagent set or reagent set described in claim 1 and Y1 is formed；The Y1 is B-P, C-P, D-P and E-P group
At；
The B-P two single stranded DNAs shown in sequence 3 in sequence table and sequence 4 form；
The C-P two single stranded DNAs shown in sequence 5 in sequence table and sequence 6 form；
The D-P two single stranded DNAs shown in sequence 7 in sequence table and sequence 8 form；
The E-P two single stranded DNAs shown in sequence 9 in sequence table and sequence 10 form.
4. detection or auxiliary detection wheat whether the composition containing haynaldia villosa 6V#4S chromosome arm, be composition B1, composition
B2 or composition B3；
Composition B1 reagent set described in claim 1 is formed with reagent needed for progress PCR amplification and/or instrument；
Composition B2 F-P as described in reagent set described in claim 1 and carry out PCR amplification needed for reagent and/
Or instrument composition；
Composition B3 reagent set described in claim 3 is formed with reagent needed for progress PCR amplification and/or instrument；
Reagent needed for the carry out PCR amplification is archaeal dna polymerase or the reagent containing archaeal dna polymerase；
Instrument needed for the carry out PCR amplification is PCR instrument.
5. haynaldia villosa molecular labeling is molecular labeling b1；
The molecular labeling b1 is that F and b is formed, and the b is that G and H is formed；
The F is DNA molecular shown in sequence 22；
The G is DNA molecular shown in sequence 23；
The H is DNA molecular shown in sequence 24.
6. haynaldia villosa molecular labeling is molecular labeling b2；
The molecular labeling b2 is the F；
The F is DNA molecular shown in sequence 22.
7. haynaldia villosa molecular labeling is molecular labeling b3；
Haynaldia villosa molecular labeling described in molecular labeling b3 haynaldia villosa molecular labeling or claim 6 as described in claim 5
It is formed with V1；The V1 is at least one of B, C, D and E；
The B is DNA molecular shown in sequence 18；
The C is DNA molecular shown in sequence 19；
The D is DNA molecular shown in sequence 20；
The E is DNA molecular shown in sequence 21.
8. detection or auxiliary detection wheat whether the method containing haynaldia villosa 6V#4S chromosome arm, be following T1):
T1) include following T11) and T12):
T11 it) respectively using the genomic DNA of wheat to be measured, reference wheat and haynaldia villosa as template, is carried out respectively with 3 kinds of primer pairs
PCR amplification obtains the wheat PCR product to be measured of 3 kinds of primer pairs, the reference wheat PCR product of 3 kinds of primer pairs and institute
State the haynaldia villosa PCR product of 3 kinds of primer pairs；The reference wheat is the wheat without haynaldia villosa 6V#4S chromosome arm, as true
Determine the reference of haynaldia villosa specific band；
T12) by the wheat PCR product to be measured of 3 kinds of primer pairs, the reference wheat PCR product of 3 kinds of primer pairs and described
The haynaldia villosa PCR product of 3 kinds of primer pairs carries out electrophoresis, determines that the wheat PCR to be measured of 3 kinds of primer pairs is produced according to electrophoresis result
In object whether the specific band containing the corresponding primer pair of haynaldia villosa, if in the wheat PCR product to be measured of 3 kinds of primer pairs extremely
Specific band containing the corresponding primer pair of haynaldia villosa in a kind of wheat PCR product to be measured of rare primer pair, the wheat to be measured
Contain or candidate contains haynaldia villosa 6V#4S chromosome arm；If be free of in the wheat PCR product to be measured of 3 kinds of primer pairs
There is the specific band of the corresponding primer pair of haynaldia villosa, the wheat to be measured is free of or candidate is without haynaldia villosa 6V#4S chromosome arm；
3 kinds of primer pairs are F-P, the G-P and the H-P described in reagent set described in claim 1；
The specific band of the corresponding primer pair of the haynaldia villosa is that the haynaldia villosa PCR product of same primer pair contains but described same
The band that the PCR product of the reference wheat of kind primer pair does not contain.
9. detection or auxiliary detection wheat whether the method containing haynaldia villosa 6V#4S chromosome arm, be following T2):
T2) include following T21) and T22):
T21) using the genomic DNA of wheat to be measured as template, PCR amplification is carried out respectively with 3 kinds of primer pairs, is obtained described 3 kinds and is drawn
The wheat PCR product to be measured of object pair；
T22) detect T21) 3 kinds of primer pairs wheat PCR product to be measured sequence, such as 3 kinds of primer pairs it is to be measured
Contain at least one in F described in the haynaldia villosa molecular labeling described in claim 5, the G and the H in wheat PCR product
Kind, the wheat to be measured contains or candidate contains haynaldia villosa 6V#4S chromosome arm；Such as the wheat PCR to be measured of 3 kinds of primer pairs
Any one of F, the G and described H described in haynaldia villosa molecular labeling described in claim 5 is free of in product or is appointed several
Kind, the wheat to be measured is free of or candidate is without haynaldia villosa 6V#4S chromosome arm；
3 kinds of primer pairs are F-P, the G-P and the H-P described in reagent set described in claim 1.
10. method according to claim 8 or claim 9, it is characterised in that: the primer annealing temperature that the PCR amplification uses for
60-64℃。
11. method according to claim 8 or claim 9, it is characterised in that: carry out PCR amplification with the F-P and the G-P
Annealing temperature is 60 DEG C, is 64 DEG C with the annealing temperature that the H-P carries out PCR amplification.
12. the application of the following Z1 or Z2 of the reagent set of claim 1 or 3:
Z1, detecting or assisting whether detection wheat contains the application in haynaldia villosa 6V#4S chromosome arm；
Z2, the application in wheat breeding.
13. following Z1 of primer pair described in claim 2 or the application of Z2:
Z1, detecting or assisting whether detection wheat contains the application in haynaldia villosa 6V#4S chromosome arm；
Z2, the application in wheat breeding.
14. following Z1 of composition as claimed in claim 4 or the application of Z2:
Z1, detecting or assisting whether detection wheat contains the application in haynaldia villosa 6V#4S chromosome arm；
Z2, the application in wheat breeding.
15. following Z1 of any the method or the application of Z2 in claim 8-11:
Z1, detecting or assisting whether detection wheat contains the application in haynaldia villosa 6V#4S chromosome arm；
Z2, the application in wheat breeding.
16. the breeding method of wheat, including with containing of being obtained according to any the method for claim 8-11 or candidate is contained
The wheat to be measured of haynaldia villosa 6V#4S chromosome arm carries out breeding as parent.
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| CN107502675B (en) * | 2017-10-16 | 2020-05-08 | 中国农业科学院作物科学研究所 | Complete set of molecular markers for detecting haynaldia villosa chromosome arms and application thereof |
| CN113528695A (en) * | 2021-06-23 | 2021-10-22 | 淮阴师范学院 | Haynaldia villosa 2VL chromosome specific RFLP-STS molecular marker and application thereof |
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| CN1737151A (en) * | 2005-07-12 | 2006-02-22 | 南京农业大学 | Wheat anti-powdery mildew gene Pm21 linked codorminant PCR marker and its usage |
| CN101121945A (en) * | 2007-06-29 | 2008-02-13 | 天津师范大学 | Special RAPD molecule marker for wheat 6VS |
| CN102807985A (en) * | 2012-08-20 | 2012-12-05 | 江苏大学 | Molecular marker 6VS-SPB with specific dasypyrum villosum 6VS chromosome and application of molecular marker |
| CN102925544A (en) * | 2012-07-03 | 2013-02-13 | 江苏里下河地区农业科学研究所 | Molecular marker for specifically tracking dasypyrumvillosum 6VS chromosome and use method thereof |
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|---|---|---|---|---|
| CN1737151A (en) * | 2005-07-12 | 2006-02-22 | 南京农业大学 | Wheat anti-powdery mildew gene Pm21 linked codorminant PCR marker and its usage |
| CN101121945A (en) * | 2007-06-29 | 2008-02-13 | 天津师范大学 | Special RAPD molecule marker for wheat 6VS |
| CN102925544A (en) * | 2012-07-03 | 2013-02-13 | 江苏里下河地区农业科学研究所 | Molecular marker for specifically tracking dasypyrumvillosum 6VS chromosome and use method thereof |
| CN102807985A (en) * | 2012-08-20 | 2012-12-05 | 江苏大学 | Molecular marker 6VS-SPB with specific dasypyrum villosum 6VS chromosome and application of molecular marker |
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