CN109880927A - Detect SNP marker primer and its application of rape BnALS1R gene - Google Patents
Detect SNP marker primer and its application of rape BnALS1R gene Download PDFInfo
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Abstract
The present invention discloses SNP marker primer and its application of detection rape BnALS1R gene.Molecular labeling provided by the invention can carry out special differentiation and detection to A the or G base in rape BnALS1R gene SNP mutational site.The application method for inventing the SNP marker provided is accurate and reliable, easy to operate, identification and assisted selection suitable for the anti-imidazolinone herbicide gene BnALS1R genotype of rape.Molecular labeling provided by the invention only needs two steps of PCR and fluorescence detection, at low cost, flux is high, high plus specificity, category filter and identification especially suitable for resistant genotypes different in breeding population.
Description
Technical field
The present invention relates to crop molecular genetic breeding technical fields, and in particular to the SNP mark of detection rape BnALS1R gene
Remember primer and its application.
Background technique
Rape (Brassica napus L.) is the important oil crops planted extensively in world wide and China
One big oil crops.Rapeseed oil acts not only as the industrial raw materials such as edible oil and lubricating oil and biodiesel.State, China
It produces rapeseed oil and accounts for 55% or more of domestic oil crops oil production, develop Rape-seed production to the national edible oil supply security tool of maintenance
There is important strategic importance (Wang Hanzhong, Chinese oil crops journal, 2018,40 (5): 613-617).However, with China's oil
Dish planting patterns is developed from traditional labor-intensive to scale, mechanization production, and it is complete that Weed infestation has become restriction rape
One of journey mechanization production, important link of light simplified cultivation.China's rape field weed type is more, quantity is big, harm weight, the Changjiang river
Basin winter rape area Weed infestation area is up to 46.9%, and northern spring rape area crop smothering area is up to 90% or more, and with dicotyledonous
Based on weeds.The winter rape area of national rapeseed area 90% is accounted for, field grass mutually can be divided into two major classes, and Paddy field after rice is grass family
Weeds and the mixed raw type of broadleaf weeds, (mitigation technology is combated a natural disaster in the production of Wang Hanzhong china rape to non-irrigated stubble Rapeseed Field based on broadleaf weeds
Beijing handbook: Scientia Agricultura Sinica technology publishing house, 2009:91-95).In production using surely kill, cover grass energy, quizalofopethyl etc.
Herbicide can effective Controlling Weeds in Rape Fields field monocotyledon weed, to mitigating Weed infestation, reduce artificial weeding labor intensity and improve oil
Dish yield plays a role.But to most broadleaf weeds, safe and efficient weeding is not yet developed in production at present
Agent.
Imidazolinone herbicide is that have strong selectivity, wide spectrum, height living using wide a kind of herbicide at present
Property, native list processing and cauline leaf are spraying before mixed soil, seedling before being broadcast, and through plant roots and leaf absorption, can prevent and kill off annual grass
Section and broadleaf weeds can also prevent and kill off perennial weeds.But such herbicide is to the general agriculture for not having anti-(resistance to) herbicidal properties
Crop itself can also generate phytotoxicity, strongly limit it using time and space, such as need crop seeding for the previous period
Just it is avoided that crops by phytotoxicity using herbicide.Chemical injury of crops can be reduced, widen by cultivating anti-(resistance to) herbicide crop varieties
The use scope of imidazolinone herbicide.Studies have shown that acetolactate synthase (acetolactate synthase, ALS) is
The target enzyme of imidazolinone herbicide, when als gene variant nucleic acid sequence causes coding protein amino acid residues site to change,
The variation of herbicide and ALS combination can be caused to generate resistance.
2004 early summer, Commercial Crop Inst., Jiangsu Prov. Academy of Agricultural Sciences Pu Huiming researcher remove spraying imidazolone type
1 plant of rapeseed plants not killed by herbicide is found in the rape and soybean crop rotation breeding field of careless agent " beans Xerox ", is moved it into
Artificial Growth case vernalization, and the maturation that bears pods until rape is bloomed.Then, by being selfed and microspores culture obtains for years
Resistance strain M9.Multiple resistance effect identification discovery, the concentration of M9 antiweed is 2~3 times of the effective herbicide concentration of herbicide,
Resistance has utility value.The author is cloned into resistant gene BnALS1R discovery, resistant gene BnALS1R and open country from M9
Raw type gene BnALS1 causes the 638th serine residue of ASL1 to be substituted by asparagine acid there are SNP at 1.Resistant gene
The China that is found to be of BnALS1R provides anti-herbicide gene with independent intellectual property rights, if can further find and resistance
The molecular labeling of gene close linkage, then can be carried out marker assisted selection, instruct field breeding, improve breeding selection efficiency.
A kind of three-primer Markers for Detection resistant gene BnALS1R genotype is disclosed in patent CN102766697B
Method.This method design of primers is complex, cumbersome, needs to do 2 PCR reactions and agarose gel electrophoresis detection, difficult
To meet the high-throughput demand of rape field breeding selection.And EB used in PCR product detection process easily causes environment
Pollution generates harm to human body.
Develop it is accurate, flexible, easy to operate detection resistant gene BnALS1R genotype molecular labeling and establish efficiently,
The coherent detection system of environmental-friendly high throughput, then it is important to promoting application of the BnALS1R gene in Hybrid breeding in commercial system to have
Meaning.
Summary of the invention
Goal of the invention: technical problem to be solved by the invention is to provide the SNP marker primers of BnALS1R gene.
There is provided SNP marker primers in detection rape BnALS1R gene generation for the present invention also technical problems to be solved
Application in terms of SNP mutation.
There is provided the detections that SNP mutation occurs for a kind of rape BnALS1R gene for the present invention also technical problems to be solved
Kit.
There is provided one kind for detecting rape BnALS1R gene the+1913rd for the present invention also technical problems to be solved
The method of base generation allelic gene typing.
Methods of genotyping the present invention is based on KASP (Kompetitive Allele-Specific PCR, KASP) is
The fluorescence signal generated during PCR is recorded and analyzed by computer, realizes and mutational site is monitored.Testing result and performance
Type consistency is high；Detection process is not necessarily to electrophoresis, has thoroughly prevented the Aerosol Pollution of PCR product, EB to the pollution of environment and right
The harm of human body.The present invention is directed to develop the KASP in resistant gene site label, quickly, resistant gene BnALS1R is accurately detected,
It lays the foundation to carry out the molecular marker assisted selection breeding of rape antiweed using resistant gene.
There is provided obtain the molecule mark with imidazolinone herbicide resistance for the present invention also technical problems to be solved
Remember assisted selection method.
There is provided the methods for identifying and increasing cross-bred rape seed purity for the last technical problems to be solved of the present invention.
Technical solution: according to early period ours the study found that the anti-imidazolinone herbicide gene BnALS1R of rape be by
Resistant Difference caused by single base mutation.The BnALS1R gene order cloned is compared with wild-type sequence, determines the SNP
At the+1913rd of rape BnALS1R gene.It is multiple that the design of two sides 50bp flanking sequence is extracted centered on candidate SNP locus
Primer sets are screened by multiple polymorphism and are obtained, and verify after many tests in multiple segregating populations, and KBnALS1R_ is marked
19681913B expanding effect is best.The label contains three primers, two specificity for the design of critical sites base difference
Primer, a universal primer.SNP marker primer provided by the invention is following specific primer sets:
(1) two specific primer:
PrimerX:5 '-TATTACATCTTTGAAAGTGCCACCAC-3 '；
PrimerY:5 '-GTTATTACATCTTTGAAAGTGCCACCAT-3 '；
(2) universal primers:
PrimerC:5 '-CAGGACCATACCTGTTGGATGTGATA-3 '.
Wherein, two specific primer sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2, described general to draw
Object is as shown in SEQ ID NO.3.
Preferably, two ends of specific primer 5 ' are separately connected different fluorescent linker sequences.It is furthermore preferred that fluorescent linker
Sequence is FAM or HEX.
The content of present invention further includes the SNP marker primer in terms of SNP mutation occurs for detection rape BnALS1R gene
Using.
The content of present invention further includes a kind of detection kit of rape BnALS1R gene generation SNP mutation, the detection examination
Agent box contains the SNP marker primer.
Wherein, the detection kit further includes KASP reaction mixture.
The content of present invention further includes a kind of for detecting the+1913rd bit base of rape BnALS1R gene generation allele
The method of parting, described detection method includes the following steps:
1) rape sample gene group DNA is extracted；
2) using rapeseed gene group DNA as template, KASP reaction detection is carried out using the SNP marker primer；
If 3) only detect the corresponding bases G of the+1913rd bit base, determine in the rape sample of detection without anti-miaow
The homozygote of oxazoline type herbicides gene BnALS1R；If only detecting the corresponding base A of the+1913rd bit base, determine
Homozygote containing anti-imidazolinone herbicide gene BnALS1R in the rape sample of detection；If the+1913rd alkali of detection site
Base is detected simultaneously by G and A, then judges the heterozygosis containing anti-imidazolinone herbicide gene BnALS1R in the rape sample of detection
Body.
Wherein, the KASP reaction detection includes PCR amplification and fluorescence detection, the PCR amplification condition are as follows: 94 DEG C
15min；94 DEG C of 20sec, 61-55 DEG C of 1min, each cycle annealing temperature reduce by 0.6 DEG C, totally 10 circulations；94 DEG C of 20sec, 55
DEG C 1min, totally 26 circulations.
The content of present invention further includes obtaining the molecular marker-assisted selection method with imidazolinone herbicide resistance, institute
The method of stating includes: to carry out genotype detection using the SNP marker primer, will contain resistant gene heterozygous single plant and circulation
Parent's backcrossing, in this way by being repeatedly returned, by genotype detection in the inbreeding population in last generation, selection contains resistant gene
Homozygous single plant can cultivate anti-imidazolinone herbicide rape.
The content of present invention further includes the method for identifying and increasing cross-bred rape seed purity, and the method is to use the step
It is rapid 1)~3) purity of obtained genotyping result statistics cross-bred rape seed, if the cross rape seed amount accounting of heterozygous
Not up to 85%, it needs through field herbicide spraying impurity elimination；If the cross rape seed amount accounting of heterozygous reaches
85% or more, it does not need through field herbicide spraying impurity elimination.
Wherein, the method identified and increase cross-bred rape seed purity is by pollinating to net cover or awarding naturally
The F of vermicelli part1Hybrid purity is identified, then increases seed purity by herbicide spraying impurity elimination in seedling stage
The utility model has the advantages that compared with prior art, the present invention has following advantages and effect:
1, KASP molecular labeling provided by the invention is functional form gene molecule marker.In breeding practice work, with mesh
The molecular labeling of gene close linkage be possible to the limitation by genetic background in application process.It is according to the present invention
SNP marker is the functional indicia based on the design of the rape mutational site functional gene ALS1, can directly reflect the anti-of plant
Property, there is no as heredity exchange and caused by mistake identification.Therefore the label can be largely avoided genetic background
Influence of the difference to testing result.
2. molecular labeling provided by the invention can carry out A the or G base in rape BnALS1R gene SNP mutational site special
Different differentiation and detection.
3, the application method for the SNP marker that invention provides is accurate and reliable, easy to operate, is suitable for the anti-imidazoline of rape
The identification of type herbicides gene BnALS1R genotype and assisted selection.
4, molecular labeling provided by the invention is in practical applications, inexpensive, high-throughput.Currently, the method for detection SNP has
PCR sequencing PCR, DNA chip, mass spectroscopic assays etc., and these methods are mostly at high cost, speed is slow.Molecular labeling provided by the invention
Only need two steps of PCR and fluorescence detection, it is at low cost, flux is high, high plus specificity, especially suitable for different in breeding population
The category filter of resistant genotype and identification.
5, molecule labelling method provided by the invention has thoroughly prevented the dirt of the Aerosol Pollution of PCR product, EB to environment
Dye and harm of the formaldehyde to human body, have it is easy, reliable, quickly, high specificity, high sensitivity, environmental-friendly remarkable advantage.
SNP marker application method provided by the invention is accurate and reliable, easy to operate, suitable for BnALS1R resistance mutation sites
Identification and assisted selection.
Detailed description of the invention
Fig. 1 KASP marks KBnALS1R_1968545B primary dcreening operation microcommunity genotyping result；
Fig. 2 marks KBnALS1R_19681913B in M9,3075R and its F1And F2Detection effect in group；
Fig. 3 marks KBnALS1R_19681913B in M9, N341 and its F1And F2Detection effect in group.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.In following embodiments method therefor unless otherwise instructed,
For conventional method documented by common molecular biology, tissue culture technique and agronomy handbook.Agents useful for same or instrument are not specified
Production firm person, being can be with conventional products that are commercially available.
LGC SNPline genotyping platform used in the present invention and its matched reagent consumptive material are purchased from Britain LGC public affairs
Department.
Embodiment 1: the SNP marker exploitation of detection Brassica Napus imidazolinone herbicide gene BnALS1R
The anti-imidazolinone herbicide gene BnALS1R of rape is to clone to obtain from resistant mutants M9, by oneself clone
BnALS1R gene order compared with wild-type sequence, determine the SNP at the+1913rd of rape BnALS1R gene.To wait
Select the sequence difference of the BnALS1R and BnALS3 that extract two sides 50bp flanking sequence centered on SNP site, and clone is combined to obtain
Design primer specific amplified BnALS1R gene order segment excludes interference of the BnALS3 gene order to detection resistant gene.Most
Eventually, we design two groups of SNP marker primers, and every group of primer is made of three primers, 3 ' end of two of them special primer
For allelic variation bases G/A, and Britain LGC (Laboratory of the Government is separately connected at 5 ' ends
Chemist) the specific FAM and HEX fluorescent linker sequence of company KASP reaction reagent.The sequence selection of universal primer will guarantee to expand
Increase segment at 60-120bp (table 1).The synthesis of all primer commissions Britain LGC company.
SNP marker primer sequence (KASP primer) of the table 1 designed for detection resistant gene BnALS1R
Embodiment 2: the foundation of the KASP molecular labeling system of the detection anti-imidazolinone herbicide gene BnALS1R of rape
Using two groups of KASP primers of above-mentioned design, M9 offspring's strain M9-2, M9-14 (Pu containing resistant gene is chosen
Hui Ming etc., the heredity of the anti-imidazolone character of rape and its application China oil crops journal, 33 (1): 15-19) and without anti-
MICMS double low restorer 3075R (public, Pu Huiming etc., Jiangsu's agriculture journal, 2010,26 (6): 1432- of property gene
1434) it is obtained with N341 (public, Pu Huiming etc., Chinese oil plant journal, 2011,33 (1): 15-19) and their hybridization
F1Generation and F2Group's single plant totally 18 rape materials carry out KASP in LGCSNPline genotyping platform and mark microcommunity
Primary dcreening operation and verifying.Specific steps are as follows:
1, the extraction of rapeseed gene group DNA:
Appropriate rape variety (strain) leaf tissue to be measured is taken, complete genome DNA (Murray M is extracted using CTAB method
G, et al., Nucleic Acids Research, 1980,8 (19): 4321-4326).With agarose electrophoresis and
Nanodrop2100 detects the quality of extracted DNA respectively, it is found that the genomic DNA extracted has reached relevant quality and wanted
It asks, i.e., agarose electrophoresis shows that DNA band is single, illustrates do not have obvious degradation disperse；Nanodrop2100 detects A260/280
Between 1.8-2.0, illustrate that DNA sample does not have protein contamination；A260/230 illustrates DNA sample salt between 2.0-2.2
Ion concentration is low；270nm does not have apparent light absorption, illustrates that DNA sample does not have phenol pollution；Dilution DNA concentration is that 20ng/ μ L is standby
With.
2, the DNA extracted using step 1 is used to detect Brassica Napus imidazoline as template using what embodiment 1 was developed
Two groups of primers of KASP label of type herbicides gene BnALS1R carry out amplification PCR, obtain amplified production.
The configuration of KASP labeled primer reaction system:
The DNA sample of 2.5 μ L extraction is added in micro reaction plate as template, KASP reaction mixture is added afterwards, instead
Answer system as follows:
2.5 μ L of template DNA
2×KASP Master mix 2.5μL
KASP Assay mix 0.07μL
PCR reaction condition are as follows: 94 DEG C of 15min；94 DEG C of 20sec, 61-55 DEG C of 1min, each cycle annealing temperature reduce by 0.6
DEG C, totally 10 recycle；94 DEG C of 20sec, 55 DEG C of 1min, totally 26 recycle.If expanding effect is undesirable to add circulation
Recycling, 3 recycle every time, at most can be three times.After the reaction was completed using scanner Pherastar to KASP reaction product
Fluorescence data reading is carried out, the result of fluorescent scanning can be converted to figure automatically.Fluorescence is detected using BMG PHERAstar instrument
Signal simultaneously checks parting situation.If parting is insufficient, continue to expand, every 3 circulations check parting situation, until parting is complete
Entirely.
2 KASP of table marks KBnALS1R_1968545B primary dcreening operation microcommunity genotyping result
Table note: R, which is represented, uses IMI class herbicide imazethapyr 45g a.i.ha-1Rapeseed plants well-grown after processing, nothing
Phytotoxicity performance；Rapeseed plants growth is heavily suppressed after S represents herbicide treatment, and final plant is dead.
3, anti-imidazolinone herbicide gene BnALS1R allelic gene typing
Allelic gene typing as a result, it has been found that, label KBnALS1R_19681913B can precisely detect anti-imidazolone type and remove
The genotype and expanding effect of careless agent gene BnALS1R is good (Fig. 1).By genotyping result and PCR product sequencing result and with
The result of the seedling stage herbicide resistance identification of field rape material compares, it has been found that label KBnALS1R_19681913B points
Type result and sequencing result, the herbicide resistance phenotypic results identified are consistent (table 2).So far, we obtain detection rapes
The KASP molecular labeling primer and detection architecture of anti-imidazolinone herbicide gene BnALS1R, specific primer are as follows:
Molecular labeling is KBnALS1R_19681913B, and polymorphic site base is G or A.Label combination includes two
Specific primer and a universal primer, wherein specific primer is respectively
TATTACATCTTTGAAAGTGCCACCAC (Prime X:SEQ ID NO.1),
GTTATTACATCTTTGAAAGTGCCACCAT (Primer Y:SEQ ID NO.2), universal primer is
CAGGACCATACCTGTTGGATGTGATA (PrimerC:SEQ ID NO.3).
It is as a result corresponding if only detecting primer Prime X by the above KASP reaction detection system using above-mentioned primer
Bases G, then determine detection rape sample in be free of anti-IMI class herbicide resistance gene BnALS1R homozygote；If only detected
To the corresponding base A of primer Primer Y, then determine in the rape sample of detection containing anti-IMI class herbicide resistance gene BnALS1R's
Homozygote；If detection site is detected simultaneously by G and A, judge in the rape sample of detection containing anti-IMI class herbicide resistance gene
The heterozygote (Fig. 1) of BnALS1R.
3 detection kit of embodiment
Augmentation detection experiment is carried out by two groups of primer pairs of embodiment 2, another set molecular labeling is
The primer combination of KBnALS1R_19681913A cannot effectively distinguish C/T genotype, and without polymorphism, so far, we are obtained
The KASP molecular labeling primer and detection architecture of detection rape BnALS1R gene resistance mutation sites, specific primer are as follows:
Specific primer: Primer X:TATTACATCTTTGAAAGTGCCACCAC, as shown in sequence SEQ IDNO.1；
Another special primer:
Primer_Y:GTTATTACATCTTTGAAAGTGCCACCAT, as shown in sequence SEQ ID NO.2；
Universal primer:
Primer_C:CAGGACCATACCTGTTGGATGTGATA, as shown in sequence SEQ ID NO.3.
Above-mentioned primer is configured to by 2: 2: 5 to the KASP Assay mix such as following table final concentration
Primer matches in 3 detection kit of table
| Primer | Primer final concentration (μM) in KASP Assay mix |
| Primer_X | 12 |
| PrimerY | 12 |
| Primer_C | 30 |
The KASP reaction detection system:
5 μ L systems:
2×KASP Master mix 2.5μL
KASP Assay mix 0.07μL
Or
10 μ L systems:
2×KASP Master mix 5μL
KASP Assay mix 0.14μL
5-10ng DNA sample to be detected is added in micro reaction plate, above-mentioned KASP reaction mixture is then added, establishes
The KASP reaction system of 5 or 10 μ L carries out PCR reaction.
By the above detection kit (KASP molecular labeling primer, KASP reaction detection system), as a result if only detected
To the corresponding bases G of primer Prime X, then determine to be free of anti-IMI class herbicide resistance gene BnALS1R's in the rape sample of detection
Homozygote；If only detecting the corresponding base A of primer Primer Y, determine to remove in the rape sample of detection containing anti-IMI class
The homozygote of careless agent gene BnALS1R；If detection site is detected simultaneously by G and A, judge in the rape sample of detection containing anti-
The heterozygote of IMI class herbicide resistance gene BnALS1R.
Embodiment 4: detection effect of the molecular labeling KBnALS1R_19681913B in segregating population
It is verifying KASP molecular labeling KBnALS1R_19681913B in F2Imidazolinone herbicide base is fought in group
Because of the detection effect of BnALS1R genotype, we are with MICMS double low restorer 3075R (public, Pu Huiming etc., Jiangsu agriculture
Industry journal, 2010,26 (6): 1432-1434) and N341 (public, Pu Huiming etc., Chinese oil plant journal, 2011,33 (1):
Be 15-19) female parent, resistance germplasm M9 be male parent rape florescence configure 2 cross combinations (3075R × M9 and N341 ×
M9), F1Selfing obtains F2Group uses molecular labeling
KBnALS1R_19681913B is to F2The genotype of the anti-herbicide gene BnALS1R of group's single plant is detected.
The specific steps such as DNA is extracted, PCR reacts, KASP detection are the same as embodiment 3.Genotypic results show in F23 are presented in group
The plant (Fig. 2 and Fig. 3) of kind genotype, the i.e. corresponding bases G of primer Primer X, then the rape single plant is without anti-IMI class
The homozygote of herbicide resistance gene BnALS1R；The corresponding base A of primer Primer Y, then the rape single plant is containing anti-IMI class weeding
The homozygote of agent gene BnALS1R；If detection site is detected simultaneously by G and A, for containing anti-IMI class weeding in the rape single plant
The heterozygote of agent gene BnALS1R.F2(3075R × M9) group is divided into 29 single plants of analysis, with the BnALS1R containing resistant gene
Homozygote single plant 7, the homozygote single plant without resistant gene BnALS1R 8, and the heterozygous of the BnALS1R containing resistant gene
Single plant 14,3 kinds of genotype meet 1: 2: 1 (χ2=0.01, P > 0.05).F2(N341 × M9) group is divided into 50 single plants of analysis,
Homozygote single plant 16 with the BnALS1R containing resistant gene, the homozygote single plant without resistant gene BnALS1R 11, and
The heterozygous single plant 23 of the BnALS1R containing resistant gene, 3 kinds of genotype meet 1: 2: (x2=0.09, P > 0.05).Two groups
Body meets monofactorial inheritance law of segregation, and label testing result is also consistent with seedling spraying herbicide identification phenotypic results.
It is indicated above that the KASP molecular labeling KBnALS1R_19681913B and detection method that are developed using the present invention can be distinguished effectively
The genotype of resistant gene BnALS1R out, and be 100% to single plant anti-herbicide gene type efficiency of selection in group.
Embodiment 5: molecular labeling KBnALS1R_19681913B assisted Selection antiweed MICMS restorer
Breeding antiweed cross-bred rape can solve the chemical weed control problem of broadleaf weed between current Rapeseed Field, save work
This is saved, and herbicide resistance can also be used in hybrid rape seed as mark property, on condition that must will resist
Herbicide resistance trait imported into MICMS restorer.It is auxiliary by marking using molecular labeling KBnALS1R_19681913B of the invention
Help selection technique can be with the MICMS restorer of the anti-IMI class herbicide of quick breeding.We are to two F1(3075R × M9 and N341
× M9) individual plant selfing obtain F2Plant in group carries out resistant gene detection, will contain homozygous resistant gene
The fertile single plant bagging of BnALS1R is selfed, and the F3 seed homozygous containing anti-IMI class herbicide resistance gene is just obtained.It eliminates simultaneously
Heterozygous and not antiweed single plant reduce the workload of breeding, eliminate and subsequent require in each separation offspring
A large amount of field trials work that herbicide spraying is identified.In addition, due to F1Maternal 3075R and N341 be with sterile thin
The restorer of cytoplasm selects in self progeny fertile single plant bagging to be selfed, so that it may which the MI CMS for bringing out anti-IMI class herbicide is extensive
Multiple system.Growth potential is strong, the maturity period is moderate resistance strain more than 50 are chosen from two 2 progeny populations at present.
This gives use molecular labeling KBnALS1R_19681913B of the invention in F2It is selected for group pure
Close the process of resistance recovery system.Certainly, breeder can also be selfed by mostly generation backcrossing, marker assisted selection, last generation
Resistant gene can be passed through recurrent selection transformation into ordinary cole by method.Basic Breeding Process is with anti-IMI class herbicide
Rape M9 is male parent, and ordinary cole is that hybridization of female parent obtains F1, then using ordinary cole as recurrent parent, before each backcrossing,
Genotype detection is carried out using molecular labeling KBnALS1R_19681913B of the invention, by the resistant gene list containing heterozygous
Strain is returned with recurrent parent.In this way by being repeatedly returned, by genotype detection in the inbreeding population in last generation, selection contains
Homozygous resistant gene single plant can cultivate Atrazine resistant Brassica napus.
Application of the 6 molecular labeling KBnALS1R_19681913B of embodiment in identification cross rape purity
Yield of rape can be increased substantially using hybrid vigour.Heterosis utilization success or not depends on two important items
Part, first is that significant hybrid vigour, second is that the hybrid seeds technology of suitable genetic cross system and highly effective and safe.China at present
The genetic cross system of cross-bred rape application mainly has Cyto-plasmtc male sterile system, genie male sterile line system and chemistry to lure
The male sterility system etc. led.Cyto-plasmtc male sterile system can generate trace-pollen, shadow because sterile line is to low-temperature sensitive in early days
Ring hybrid purity.Genie male sterile line system increases miscellaneous because that must pull out fertile plant during sterile line propagation or the production of hybrid seeds
The difficulty and cost of kind production, and artificial removal's technical requirements are high, inevitably have omission, also will affect purity of hybrid.Chemistry
The male sterility of induction is that the bud for handling normal self-mating system using gametocide or chemical hybridizing agent (CHA) induces one generated
Kind of male sterility, the highest attention by rapeseed breeding man in recent years, have become one that rape heterosis utilizes it is important
Approach.During the chemical emasculation production of hybrid seeds, if maternal gametocide is not thorough enough, self-fertility is caused, it is pure also to will affect cenospecies
Degree.Therefore hybrid purity is an important factor for restricting cross-bred rape yield potential.
Pre-stage test show we by herbicide resistance trait transformation into restorer material, by cenospecies seedling stage
The selfing strain of removal sterile line can increase hybrid F while spraying IMI class herbicide removal weeds in field1Purity.The present invention provides
A kind of new strategy, can first pass through high-throughput molecular labeling KBnALS1R_19681913B classifying method of the invention, to miscellaneous
It hands over rape seed to carry out Purity in advance, cross rape state quality standard GB4407.2-2008 is reached to seed purity
It is required that, there is no need to pass through the impurity elimination of seedling spraying herbicide.It is counted according to genotyping result obtained in 1 step 3 of examples detailed above
The purity of cross-bred rape seed, if the cross rape seed amount accounting of heterozygous is not up to 85%, it is necessary to pass through field
Herbicide spraying impurity elimination；If the cross rape seed amount accounting of heterozygous reaches 85%, it may not be necessary to be sprayed by field
Spraying herbicide impurity elimination.We by resistant gene BnALS1R transformation into MICMS restorer, under 3 kinds of different pollination conditions and not
Educating is hybridization, obtains F1Hybrid, by carrying out purity to seed using molecular labeling KBnALS1R_19681913B of the invention
Identification, the F under the conditions of the pollination of discovery net cover and spontaneous pollination185% national standard is not achieved in hybrid purity, then
13% and 16% (table is respectively increased by the herbicide imazethapyr impurity elimination of seedling spraying IMI class, relative to control purity in we
4).Before this, the purity of cross-bred rape seed, it is main to be reflected by field plot plantation and DNA fingerprinting technology
It is fixed.The former, is protected from environmental larger, and qualification time is long；The latter then needs to develop more molecular labeling, and each
Sample requires the Marker Identification of all exploitations, larger workload.
Table 4 marks KBnALS1R_19681913B identification of seed purity and herbicide spraying to F1The raising of hybrid purity
The foregoing is merely presently preferred embodiments of the present invention and oneself, be not intended to limit the invention, it is all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>SNP marker primer and its application of rape BnALS1R gene are detected
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence (Primer X)
<400> 1
tattacatct ttgaaagtgc caccac 26
<210> 2
<211> 28
<212> DNA
<213>artificial sequence (Primer Y)
<400> 2
gttattacat ctttgaaagt gccaccat 28
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Primer C)
<400> 3
caggaccata cctgttggat gtgata 26
Claims (10)
- The SNP marker primer of 1.BnALS1R gene, which is characterized in that the SNP marker primer include two specific primers and One universal primer, two specific primer sequences are described general to draw as shown in SEQ ID NO.1 and SEQ ID NO.2 Object is as shown in SEQ ID NO.3.
- 2. SNP marker primer according to claim 1, which is characterized in that the end two specific primer 5' connects respectively Connect different fluorescent linker sequences.
- 3. application of the SNP marker primer as claimed in claim 1 or 2 in terms of SNP mutation occurs for detection rape BnALS1R gene.
- 4. the detection kit that SNP mutation occurs for a kind of rape BnALS1R gene, which is characterized in that the detection kit contains SNP marker primer described in having the right to require 1 or 2.
- 5. detection kit according to claim 4, which is characterized in that the detection kit further includes that KASP reaction is mixed Close liquid.
- 6. a kind of method that allelic gene typing occurs for detecting the+1913rd bit base of rape BnALS1R gene, feature exist In described detection method includes the following steps:1) rape sample gene group DNA is extracted；2) using rapeseed gene group DNA as template, KASP reaction inspection is carried out using SNP marker primer described in as claimed in claim 1 or 22 It surveys；If 3) only detect the corresponding bases G of the+1913rd bit base, determine in the rape sample of detection without anti-imidazoline The homozygote of type herbicides gene BnALS1R；If only detecting the corresponding base A of the+1913rd bit base, determine to detect Rape sample in the homozygote containing anti-imidazolinone herbicide gene BnALS1R；If the+1913rd bit base of detection site is same When detect G and A, then judge detection rape sample in the heterozygote containing anti-imidazolinone herbicide gene BnALS1R.
- 7. according to the method described in claim 6, it is characterized in that, the KASP reaction detection includes PCR amplification and fluorescence inspection It surveys, the PCR amplification condition are as follows: 94 DEG C of 15min；94 DEG C of 20sec, 61-55 DEG C of 1min, each cycle annealing temperature reduce 0.6 DEG C, totally 10 recycle；94 DEG C of 20sec, 55 DEG C of 1min, totally 26 recycle.
- 8. obtaining the molecular marker-assisted selection method with imidazolinone herbicide resistance, which is characterized in that the method Include: that genotype detection is carried out using SNP marker primer of any of claims 1 or 2, resistant gene heterozygous single plant will be contained It is returned with recurrent parent, in this way by being repeatedly returned, by genotype detection in the inbreeding population in last generation, selection is containing anti- Property gene pure type single plant can cultivate anti-imidazolinone herbicide rape.
- 9. the method for identifying and increasing cross-bred rape seed purity, which is characterized in that the method is using described in claim 6 Step 1) ~ 3) obtain genotyping result statistics cross-bred rape seed purity, if the cross rape seed amount of heterozygous accounts for Than being not up to 85%, need through field herbicide spraying impurity elimination；If the cross rape seed amount accounting of heterozygous reaches 85% or more, it does not need through field herbicide spraying impurity elimination.
- 10. identification according to claim 9 and the method for increasing cross-bred rape seed purity, which is characterized in that the mirror The method for determining and increasing cross-bred rape seed purity is by the F1 hybrid purity to net cover pollination or spontaneous pollination condition It is identified, seed purity is then increased by herbicide spraying impurity elimination in seedling stage.
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| CN114540347A (en) * | 2022-03-15 | 2022-05-27 | 华智生物技术有限公司 | KASP marker primer and kit for identifying imidazolinone herbicide-resistant oilseed rape and application of KASP marker primer and kit |
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