CN105861496B - A kind of molecular labeling and its application with balsam pear mildew-resistance gene close linkage - Google Patents
A kind of molecular labeling and its application with balsam pear mildew-resistance gene close linkage Download PDFInfo
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Abstract
The present invention is to build balsam pear heredity segregating population with disease-resistant balsam pear MC18 and susceptible balsam pear MC105 hybridization, using genome deep sequencing technology is simplified, 91,856 SLAF labels is obtained altogether, screens 29 difference marks closely related with balsam pear powder mildew resistance.Using molecular labeling of the invention marked as SLAF4542, using tetra-primer ARMS-PCR PCR, in the identification of balsam pear powder mildew resistance, two specific fragments of 356bp and 326bp are amplified in height sense and the identification of susceptible balsam pear, and the specific fragment of 326bp is only amplified in anti-balsam pear identification high.This is marked at F2 and reaches more than 98% for the recall rate in segregating population, molecular marker assisted selection can be carried out to the mildew-resistance gene in balsam pear breeding material in seedling stage powdery mildew non-premorbid, the effective deficiency for overcoming conventional breeding, simplify system of selection and improve breeding efficiency, accelerate the process of balsam pear breeding for disease resistance.
Description
Technical field
The invention belongs to plant molecular genetics technical field, and in particular to one kind closely connects with balsam pear mildew-resistance gene
The application of the molecular labeling of lock and its molecular labeling in balsam pear mildew-resistance breeding.
Background technology
Balsam pear (Momordica charantia L.) is the annual climbing herb plant of Curcurbitaceae Momordica, is had higher
Nutritive value and dietotherapy effect.In recent years, balsam pear cultivated area is continuously increased, it has also become the staple vegetable kind of " south vegetable north transportation "
And specialty industries.Powdery mildew is a kind of worldwide disease that melon vegetables occurs extensively, can be occurred from seedling stage to harvest time.Closely
Nian Lai, increase and large-scale development with melon vegetables cultivated area, under powdery mildew has turned into and causes the underproduction and fruit quality
Drop, hinders one of Major Diseases of green production.Such as in Guangxi balsam pear planting base Wu Tang towns, year nearly 20,000 mu of cultivated area, year
Yield is more than 45,000,000 kilograms, and the incidence of disease of powdery mildew is up to more than 90%, because of production loss nearly 1000 caused by powdery mildew
Ten thousand kilograms, economic well-being of workers and staff is lost up to 15,000,000 yuan, causes serious economic loss.
Powdery mildew can occur in each period of balsam pear field growing, and in seedling stage, morbidity can cause balsam pear growth potential to subtract
It is weak, hypoevolutism；In florescence, morbidity can cause balsam pear underproduction 20-40%；In the morbidity of knot melon phase, plant leaf can be caused withered
Huang, fruit growth is slow, and plant early ageing has a strong impact on the quality and yield of fruit.Peasant household consumes during powdery mildew is prevented and treated
Take substantial amounts of labour and the means of agricultural production, the agricultural chemicals for excessively using all causes greatly infringement to product quality and environment, therefore, training
It is a most safe, economic, effective approach for defending powdery mildew to bring out disease-resistant variety.
Because Powdery Mildew can only be preserved using live body, occurring degree easily it is affected by environment the features such as, give disease resistance mirror
Surely very big inconvenience is brought, traditional breeding for disease resistance cycle is long, it is necessary to a large amount of and many generation configuration cross combinations and inoculation powdery mildew observation,
Seriously hinder melon vegetables mildew-resistance breed breeding process.
With developing rapidly for Protocols in Molecular Biology, molecular breeding technology greatly accelerates the process of breeding for disease resistance, shows
Work shortens the cycle of disease-resistant variety seed selection.DNA molecular marker technology has polymorphism high, easy to detect, quick, accurate because of it
The features such as, extremely important effect has been played in disease-resistant crops genetic breeding.SLAF-seq(specific length
Amplified fragment sequencing) technology developed by high throughput sequencing technologies.Entered by bioinformatics
Row experimental program system design, builds SLAF-seq libraries, screens special length DNA fragmentation, is obtained with high throughput sequencing technologies
Magnanimity sequence, data analysis comparison is carried out using software, obtains a large amount of specific DNA fragments, and then develop spy according to its sequence
Different molecular labeling.SLAF-seq technologies have the outstanding advantages such as flux high, accuracy is high, low cost, cycle is short, according to its sequencing knot
Fruit can directly develop a large amount of specific molecular markers.The technology can be used for haplotype collection of illustrative plates, genetic map, relevance collection of illustrative plates, polymorphic
The structure of property collection of illustrative plates, for molecular breeding, phyletic evolution, germplasm identification provide important technology guarantee.
The present invention utilizes SLAF-seq technological development a collection of high specificity, stability cucumber powdery mildew resistance high special
Molecular labeling, carries out molecular marker assisted selection and provides important foundation to import resistance excellent genes in breeding.
The content of the invention
To solve the above problems, the invention provides a kind of Novel bitter gourd SNP marker, can not sent out in seedling stage powdery mildew
Molecular marker assisted selection is carried out to the mildew-resistance gene in balsam pear breeding material before disease, conventional breeding is effectively overcome
Deficiency, simplifies system of selection and improves breeding efficiency, accelerates the process of balsam pear breeding for disease resistance.
In order to realize foregoing invention purpose, the present invention uses following technical scheme：
A kind of molecular labeling with balsam pear mildew-resistance gene close linkage, described molecular labeling marked as
SLAF4542, it has the sequence as shown in Seq ID NO.1.
Preferably, described molecular labeling is by primer pair Primer Forward inner and Primer Reverse
What inner, primer pair Primer Forward outer and Primer Reverse outer amplification were obtained.
Preferably, described primer pair Primer Forward inner, Primer Reverse inner, Primer
Forward outer and Primer Reverse outer have the sequence shown in Seq ID NO.2-5 respectively.
Present invention also offers a kind of PCR detection kit of balsam pear mildew-resistance gene, described PCR detection kit
Reaction system totally 10 μ L：The template DNA of 1.0 μ L concentration 30ng/ μ L；1 μm of μ of Primer Forward outer 1.0 of ol.L-1
L；1 μm of μ L of Primer Reverse outer 1.0 of ol.L-1；10 μm of μ of Primer Forward inner 1.0 of ol.L-1
L；10 μm of μ L of Primer Reverse inner 1.0 of ol.L-1；10×buffer 1.0μL；The dNTP of 2m mol.L-1
1.0μL；50 μm of MgCl of ol.L-120.4μL；The μ L of Taq enzyme 0.1；ddH2The μ L of O 2.5, it is characterised in that described primer pair
Primer Forward inner and Primer Reverse inner, primer pair Primer Forward outer and Primer
Reverse outer have the sequence shown in Seq ID NO.2-5 respectively.
Present invention also offers it is described with balsam pear mildew-resistance gene close linkage molecular labeling in balsam pear powdery mildew
Application in Resistance Identification, comprises the following steps：
(1) balsam pear genomic DNA to be detected is extracted；
(2) with balsam pear genome to be detected as template, using the primer pair described in right 3, the reactant described in right 4
System, carries out pcr amplification reaction；
(3) through electrophoresis, such as band amplifies the specific fragment of 326bp, then be mildew-resistance balsam pear high；As band is amplified
Two specific fragments of 356bp and 326bp, then be height sense powdery mildew balsam pear.
Compared with prior art, the present invention has the advantages that：
The present invention is to build balsam pear heredity segregating population with disease-resistant balsam pear MC18 and susceptible balsam pear MC105 hybridization.The present invention
Using genome deep sequencing technology is simplified, 91,856 SLAF labels are obtained altogether, screen 29 with balsam pear powder mildew resistance
Closely related difference mark.Wherein it is obstructed using the amplification of four primers prominent marked as SLAF4542 using described molecular labeling
Change system PCR, in the identification of balsam pear powder mildew resistance, the two of 356bp and 326bp is amplified in height sense and the identification of susceptible balsam pear
Bar specific fragment, only amplifies the specific fragment of 326bp in anti-balsam pear identification high.This is marked at F2 for the inspection in segregating population
Extracting rate reaches more than 98%, can carry out molecule to the mildew-resistance gene in balsam pear breeding material in the non-premorbid of seedling stage powdery mildew
Marker assisted selection, effectively overcomes the deficiency of conventional breeding, simplifies system of selection and improves breeding efficiency, accelerates balsam pear
The process of breeding for disease resistance.
Brief description of the drawings
Fig. 1 is balsam pear simplification gene order-checking mass value distribution map in the embodiment of the present invention；
Wherein, abscissa is the base positions of reads, and ordinate is the mass value of single base.Preceding 80bp is sequenced for both-end
The first end of sequence is sequenced the quality Distribution value of reads, and rear 80bp is the quality Distribution value that the other end is sequenced reads.Each bp
Each base of all reads of sequencing is represented, each quality color of same position is got over to deeply feel and shows this mass value in data
The ratio of obtaining is higher.
Fig. 2 is balsam pear simplification gene order-checking base contentses distribution map in the embodiment of the present invention；
Wherein, abscissa is the base positions of reads, and ordinate is the ratio shared by base；Different colours represent difference
Base type, green represents base A, and blueness represents base T, and red represents base C, and orange to represent bases G, grey is represented to be surveyed
The base N that can not recognized in sequence.The base distribution of Reads is sequenced for the first end of both-end sequencing sequence for preceding 80bp, and rear 80bp is
The other end is sequenced the base distribution of reads.Each bp represents each base of sequencing, and a such as bp is to represent all surveys of the project
Sequence reads is in first distribution situation of A, T, G, C, N of base.
Fig. 3 is that balsam pear powdery mildew endangers field symptom；
Fig. 4 is identification powdery mildew disease index in the embodiment of the present invention；
Wherein, a disease indexs are 0 grade (whole blade is without amyloid plaque)；B, c disease index (have a small number of amyloid plaques, but powder for 1 grade
Spot area less than blade area 1/3)；D disease indexs are 2 grades (amyloid plaque increases the 1/3-2/3 for accounting for full leaf area)；The e state of an illness refers to
Number for 3 grades (amyloid plaque is gradually connected in flakes, occupied area more than leaf area 2/3)；F, g disease index are 4 grades, and (amyloid plaque is paved with whole
Blade is opened, blade is touched, white powder shakes)；H disease indexs are 5 grades, and (amyloid plaque is covered with whole blade, and blade starts yellow, gradually withered
Extremely).
Fig. 5 is anti-, high sense balsam pear genome dna electrophoresis figure high in the embodiment of the present invention；
Wherein, swimming lane 1 is mildew-resistance balsam pear DNA high；Swimming lane 2 is mildew-resistance balsam pear DNA high；Swimming lane 3 is Gao Kangbai
Powder suffering of illness melon DNA；Swimming lane 4 is mildew-resistance balsam pear DNA high；Swimming lane 5 is sense powdery mildew balsam pear DNA high；Swimming lane 6 is sense white powder high
Suffering of illness melon DNA；Swimming lane 7 is sense powdery mildew balsam pear DNA high；Swimming lane 8 is sense powdery mildew balsam pear DNA high.
Fig. 6 be the embodiment of the present invention in SLAF4542 molecular labelings in parent material and its F2 for the amplification figure in individual plant
Spectrum；
Wherein, swimming lane 1 is DNAmarker 1000；Swimming lane 2 is male parent MC18 (356bp)；The female parent of swimming lane 3 MC105
(356bp, 326bp)；Swimming lane 4 is mildew-resistance balsam pear HR1 (356bp) high；Swimming lane 5 is mildew-resistance balsam pear HR3 high
(356bp)；Swimming lane 6 is mildew-resistance balsam pear HR17 (356bp) high；Swimming lane 7 is mildew-resistance balsam pear HR30 (356bp) high；Swimming
Road 8 is sense white powder suffering of illness HS4 (356bp, 326bp) high, and swimming lane 9 is sense white powder suffering of illness HS7 (356bp, 326bp) high, swimming lane 10
It is height sense white powder suffering of illness HS18 (356bp, 326bp), swimming lane 11 is sense white powder suffering of illness HS31 (356bp, 326bp) high.
Specific embodiment
With reference to specific embodiment, further details of elaboration is made to the present invention, but embodiments of the present invention are not
It is confined to the scope that embodiment is represented.These embodiments are merely to illustrate the present invention, not for limitation the scope of the present invention.This
Outward, after present disclosure is read, those skilled in the art can various modifications may be made to the present invention, and these equivalent variations are same
Sample falls within appended claims limited range of the present invention.
Experimental technique used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent for using etc., unless otherwise specified, commercially obtain.
Embodiment 1：
In the embodiment of the present invention with disease-resistant material MC18 as male parent, susceptible material MC105 for female parent, according to its be inoculated with white powder
Anti-/sense performance after being ill, using BSA methods in the F2 colonies for building, the family group of 30 mildew-resistances of picking and sense powdery mildew
Into anti-/ sense powdery mildew pond.Material is planted in Guangxi Academy of Agricultural Sciences Vegetable Research Institute scientific base.By parent MC18, MC105
And F2 colonies strain is in temperature-controlled, the intelligent temperature indoor pot of humidity.In the heart stage of one leaf of balsam pear seedling one, connect using friction
The method of kind, inoculum density is 1 × 104-1×106Individual spore/mL.Observation in every three days, incidence of record, record altogether after inoculation
15 days.Then according to the phenotype result for resisting/feeling powdery mildew, high anti-and high each 30 of the plant felt of picking constitutes anti-/ sense white powder
Sick pond.
The implementation case utilizes 1580 plants of F2Colony, identifies powdery mildew disease index.The authentication method of disease index is as follows
(referring to Fig. 4)：
0 grade：Whole blade is without amyloid plaque；
1 grade：There are a small number of amyloid plaques, but amyloid plaque area less than the 1/3 of blade area；
2 grades：Amyloid plaque increases the 1/3-2/3 for accounting for full leaf area；
3 grades：Amyloid plaque is gradually connected in flakes, and occupied area exceedes the 2/3 of leaf area；
4 grades：Amyloid plaque is paved with whole blade, touches blade, and white powder shakes；
5 grades：Amyloid plaque is covered with whole blade, and blade starts yellow, gradually withered.
Calculate disease index：
Formula is：DI=∑s (sini)/5N × 100
DI=disease indexs, si=onset grades, the number of sheets of the corresponding onset grades of ni=, each level of i=severity Scalings
Not, N=investigation total number of sheets.
The balsam pear tender leaf of the heart stage of two leaf one is taken, its genomic DNA is extracted using the CTAB methods of improvement, be diluted to 60ng/ μ L,
Through 1% agarose electrophoresis detectable concentration.Network analysis is carried out using digestion forecasting software SLAF_Predict, is contained according to its GC
Amount, repetitive sequence and genetic traits etc. determine digestion scheme, cut glue scope and sequencing amount, to ensure the density and of molecular labeling
Even property.
According to selected most suitable digestion scheme, each sample genomic DNA to detecting qualified carries out digestion respectively.To obtaining
Endonuclease bamhi (SLAF labels) carry out 3 ' ends plus A treatment, connect Dual-index sequence measuring joints, PCR amplifications, purifying, sample mixing,
Cut glue and choose purpose fragment, be sequenced with IlluminaHiSeqTM 2500 after library quality inspection is qualified.
The initial data that sequencing is obtained is identified using Dual-index, obtains the reads of each sample.Filtering is surveyed
After the joint of sequence reads, the assessment of sequencing quality and data volume is carried out.By Control data assessment digesting efficiencies, sentenced with this
The accuracy and validity of disconnected experimentation.Method by being clustered between reads, develops SLAF labels in parent and mixed pond,
There is the SLAF labels of polymorphism in parent in searching.
The embodiment of the present invention obtains 91,856 SLAF labels altogether, and the wherein sequencing depth of male parent is 19.01x, maternal
Sequencing depth is 24.64x, and polymorphic sex ratio is 5.99%.Analysis is associated to polymorphism SLAF labels, screen 29 with
The associated Marker of objective trait.
For 29 Marker being associated with objective trait are screened, functional verification and analysis are carried out.
Using tetra-primer ARMS-PCR PCR (Tetra-primer amplification refractory
Mutation system PCR, Tetra-primerARMS PCR) carry out design of primers and reaction system optimization.
Tetra-primerARMS PCR are a kind of SNP calls grown up on the basis of regular-PCR
Technology, ranges ApoE gene method.This method solve that operated in accordance with conventional methods is cumbersome, the low problem of parting flux,
With easy to operate, parting it is quick, it is low-cost the features such as, be widely used to the research in the fields such as medical science, but utilize the party
Method does not inquire the documents and materials of correlation also at present to the research report of balsam pear.
The embodiment of the present invention designs 12 pairs of Tetra-primerARMS PCR primers altogether, wherein using described molecular labeling
Marked as SLAF4542, two specific fragments of 356bp and 326bp are amplified in the identification of disease-resistant balsam pear, felt and susceptible in height
The specific fragment of 326bp is only amplified in balsam pear identification.This is marked at F2 and reaches more than 98% for the recall rate in segregating population,
Molecular marker assisted selection can be carried out to the mildew-resistance gene in balsam pear breeding material in seedling stage powdery mildew non-premorbid.
Balsam pear genomic DNA is extracted using the CTAB methods of improvement, extraction step is as follows：
(1) the fresh balsam pear tender leafs of 2g are weighed, adds 1% PVP quickly to be pulverized under liquid nitrogen, it is stand-by；
(2) ground material is quickly adding into the 2%CTAB extract solutions (2%CTAB that 65 DEG C of 500 μ L are fully warmed-up
The formula of extract solution is：100mM Tris-HCL, pH 8.0,20mM EDTA, 1.4M NaCL) in, and add 20 μ L β-sulfydryl
Ethanol, fully vibration are mixed, and the water-bath 2h at 65 DEG C, period turns upside down mixing centrifuge tube every 10min；
(3) room temperature is cooled to after taking out, isometric chloroform-isoamyl alcohol is added, mixing of turning upside down emulsifies it, 4 DEG C,
12 000r/min are centrifuged 10min；
(4) Aspirate supernatant adds the isopropanol of isometric precooling in another clean centrifuge tube, overturns and mixes, 1-
Occurs white flock precipitate after 2min；
(5) precipitation is transferred in the centrifuge tube for filling 75% ethanol after 4000r/min centrifugations and is washed 2 times, add TE molten
Solution precipitation；
(6) add 5 μ LRNase A (about 40-100 μ g) that RNAs of the 30min to contain in removing sample is digested in 37 DEG C；
(7) isometric chloroform-isoamyl alcohol is added, 10 000r/min centrifugations 10min after mixing takes supernatant and repeats to take out
Carry 2 times；
(8) NaCL of 400 μ L 5mol/L is added, 30min is stood after mixing on ice, then 10000r/min centrifugation
10min, takes supernatant；
(9) 2 times of absolute ethyl alcohols of volume are added, 20min are placed for 4 DEG C after mixing, then 6 000r/min centrifugations 10min,
Supernatant is outwelled, is dried；
(10) washed 1 time with absolute ethyl alcohol after precipitation being washed into 2 times with 75% ethanol, add 100 μ L TE to buffer after draining
Liquid dissolves, and is stored in -20 DEG C of refrigerators.
The balsam pear genomic DNA of extraction is carried out into concentration and quality testing, under 100V constant voltages, 1% Ago-Gel
Electrophoresis 30 minutes, detects DNA mass.Use spectrophotometry DNA purity.
DNA absorbs strongly at 260nm, and the value of OD260/OD280 illustrates that purification degree preferably, is less than between 1.7-1.9
1.7, illustrate to remain more protein in the DNA for extracting, can continue to extract with phenol, chloroform；If being more than 2.0, RNA has been illustrated
Pollution or DNA fracture.
Balsam pear powder mildew resistance identifies method for quick, the base sequence containing SLAF4542 in PCR reaction systems
Primer pair Primer Forward inner and Primer Reverse inner, Primer Forward outer and Primer
Reverse outer。
Primer Forward inner：5’-TATGCGCGCCAAACCAAATA-3’；
Primer Reverse inner：5’-GCTTCTAGTGCTTTATATGAGTGTCTACC-3’；
Primer Forward outer：5’-CCACTGGAATTCCCACCTACAC-3’；
Primer Reverse outer：5’-CGTCAATCTGAAATCGACTAAAGTACC-3’.
In the embodiment of the present invention, the reaction system and amplification program of PCR are as follows respectively：
Reaction system totally 10 μ L：The template DNA of 1.0 μ L concentration 30ng/ μ L；1μmol.L-1Primer outer F 1.0
μL；1μmol.L-1The μ L of Primer outer R 1.0；10μmol.L-1The μ L of Primer inner F 1.0；10μmol.L-1
The μ L of Primer inner R 1.0；10×buffer 1.0μL；2m mol.L-1The μ L of dNTP 1.0；50μmol.L-1's
MgCl20.4μL；The μ L of Taq enzyme 0.1；ddH2O 2.5μL。
Amplification program：94 DEG C of predegeneration 5min；94 DEG C of denaturation 45s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min；Totally 35 are followed
Ring；72 DEG C of extension 7min；15 DEG C save backup.
PCR primer agarose gel electrophoresis detection method in the embodiment of the present invention, gum concentration is 2%, 1 μ L loading
Buffer+4 μ L PCR primers, 100V voltages, electrophoresis 1h 30min.
As shown in Figure 5, Figure 6, using molecular labeling SLAF4542 of the invention, using tetra-primer ARMS-PCR
PCR, in the identification of balsam pear powder mildew resistance, identifies the Liang Tiaote for amplifying 356bp and 326bp in height sense and susceptible balsam pear
Heteroleptic, only amplifies the specific fragment of 326bp in anti-balsam pear identification high.
In sum, molecular labeling of the invention reaches more than 98% in F2 for the recall rate in segregating population, can be in seedling
The non-premorbid of phase powdery mildew carries out molecular marker assisted selection to the mildew-resistance gene in balsam pear breeding material, effectively overcomes
The deficiency of conventional breeding, simplifies system of selection and improves breeding efficiency, accelerates the process of balsam pear breeding for disease resistance.
Claims (3)
1. a kind of molecular labeling of and balsam pear mildew-resistance gene close linkage, it is characterised in that：The mark of described molecular labeling
Number be SLAF4542, it has the sequence as shown in Seq ID NO.1；Described molecular labeling is by primer pair Primer
Forward inner and Primer Reverse inner, primer pair Primer Forward outer and Primer
Reverse outer amplifications are obtained；Described primer pair Primer Forward inner and Primer Reverse
Inner, primer pair Primer Forward outer and Primer Reverse outer have Seq ID NO.2-5 institutes respectively
The sequence shown.
2. a kind of PCR detection kit of balsam pear mildew-resistance gene, described PCR detection kit reaction system totally 10 μ L：
The template DNA of 1.0 μ L concentration 30ng/ μ L；1 μm of μ L of Primer Forward outer 1.0 of ol.L-1；1 μm of ol.L-1's
Primer Reverse outer 1.0μL；10 μm of μ L of Primer Forward inner 1.0 of ol.L-1；10μmol.L-1
The μ L of Primer Reverse inner 1.0；10×buffer 1.0μL；The μ L of dNTP 1.0 of 2m mol.L-1；50μ
The MgCl of mol.L-120.4μL；The μ L of Taq enzyme 0.1；ddH2The μ L of O 2.5, it is characterised in that described primer pair Primer
Forward inner and Primer Reverse inner, primer pair Primer Forward outer and Primer
Reverse outer have the sequence shown in Seq ID NO.2-5 respectively.
3. the molecular labeling of according to claim 1 and balsam pear mildew-resistance gene close linkage is in balsam pear powder mildew resistance
Application in identification, it is characterised in that comprise the following steps：
(1) balsam pear genomic DNA to be detected is extracted；
(2) with balsam pear genome to be detected as template, using the primer pair described in right 3, the reaction system described in right 4, enter
Performing PCR amplified reaction；
(3) through electrophoresis, such as band amplifies the specific fragment of 326bp, then be mildew-resistance balsam pear high；As band is amplified
Two specific fragments of 356bp and 326bp, then be height sense powdery mildew balsam pear.
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| 基于简化基因组深度测序技术的黄瓜白粉病主效抗性基因精细定位及候选基因挖掘;张鹏;《中国园艺学会黄瓜分会第四届年会论文摘要集》;20131107;全文相关 * |
| 苦瓜抗白粉病SRAP标记筛选的PCR体系优化与验证;米军红;《南方农业学报》;20130615;第44卷(第6期);全文相关 * |
| 苦瓜抗白粉病基因的分子标记筛选及初步定位;米军红;《中国优秀硕士学位论文全文数据库 农业科技辑》;20140315(第2014年3期);摘要，正文第9、12-13、15-16、20-21、35-39页 * |
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