CN109536631A

CN109536631A - Multiple PCR method that is a kind of while detecting tomato disease-resistant gene Sw-5 and I-2

Info

Publication number: CN109536631A
Application number: CN201811553843.9A
Authority: CN
Inventors: 宋建军; 孙功; 李振侠
Original assignee: Hebei University of Science and Technology
Current assignee: Hebei University of Science and Technology
Priority date: 2018-12-19
Filing date: 2018-12-19
Publication date: 2019-03-29

Abstract

Tomato disease-resistant gene is detected simultaneously the present invention relates to a kind ofSw‑ 5WithI‑2Multiple PCR method comprising following steps, (1) design of primers；(2) CTAB method is used, tomato leaf DNA to be detected is extracted from tomato leaf；(3) DNA obtained is extracted as template using step (2), utilizeI‑2WithSw‑5Primer multiplexed PCR amplification is carried out to sample to be tested by the multi-PRC reaction system of foundation；(4) PCR product is subjected to 1.5% agarose gel electrophoresis, is imaged using gel imaging system.The present invention can efficiently and accurately identify the disease-resistant heterozygosis of tomato, homozygous genotype and susceptible genotype.Using the present invention, can save can effectively assist tomato breeding for disease-resistance to the time of two kinds of disease-resistant genes of tomato while detection and cost, testing result.

Description

Multiple PCR method that is a kind of while detecting tomato disease-resistant gene Sw-5 and I-2

Technical field

The present invention relates to technical field of biological, and in particular to a kind of to detect tomato disease-resistant gene simultaneouslySw-5WithI-2 Multiple PCR method.

Background technique

Tomato (Lycopersicon. esculentumMill) it is one of most important vegetable crop in the world, at me State plants various regions extensively.Tomato accounts for critical role in the dietary structure of people with delicious, full of nutrition famous.However, tomato Disease species are various, and more serious tomato disease occurs in the world just more than 40 kinds, and in China, also having of generally occurring is ten several. In recent years, with the diversification of the increase of cultivated area and planting form, tomato in China disease is got worse.

Tomato spotted wilf virus disease be by bunyaviridae (Bunyaviridae)Tospovirus (Tospovirus) tomato spotted wilf virus (Tomato spotted wilt virus, TSWV) caused by.It is passed by thrips It broadcasts.Tomato spotted wilf virus can also be propagated by approach such as juice frictional inoculation, graftings, but daily contact and seed is not The virus can be propagated.The gene of the anti-tomato spotted wilf virus identified on different tomato materials at present hasSw-1 ^a、Sw-1 ^b、Sw- 2、Sw-3、Sw-4、Sw-5、Sw-6WithSw-7.WhereinSw-5It is considered as anti-tomato spotted wilf virus disease in tomato breeding practice Major gene resistance, be study at present it is more and in business breeding using most resistant genes.

Tomato wilt (Fusarium oxysporumf. sp. lycopersici) sent out from nineteen fifties Since existing, various regions all over the world, it has also become the major issue in tomato production.Stevens and Rick hair in 1986 The biological strain of existing tomato wilt has 3, and the disease-resistant variety being bred as both at home and abroad at present mainly contains disease-resistant geneI-2, it is disease-resistant GeneI-3Or contain disease-resistant gene simultaneouslyI-1WithI-2。Due toI-2Gene simultaneously anti-blight biological strain 1 and 2, kind Compare in eggplant breeding and is paid attention to.

It is reported currently, multiple PCR technique has part in tomato breeding for disease-resistance, but identifies anti-tomato spotted wilt base simultaneously CauseSw-5With anti-tomato wilt geneI-2Multi-PRC reaction system has not been reported.

Summary of the invention

The object of the present invention is to provide detect tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR technique, i.e., it is a kind of same When detect tomato disease-resistant geneSw-5WithI-2Multiple PCR method, can be used in Rapid identification tomato material whether contain simultaneously There is the disease-resistant gene of tomato spotted wilf virusSw-5With the disease-resistant gene of tomato wiltI-2。

To achieve the above object, preparation method of the present invention includes the following steps that it includes the following steps,

(1) design of primers,

I-2Primer are as follows:

I-2 F is SEQ ID No.1:5 '-CAAGGAACTGCGTCTGTCTG-3 '；

I-2 R is SEQ ID No.2:5 '-ATGAGCAATTTGTGGCCATG-3 '；

Sw-5Primer are as follows:

Sw-5 F is SEQ ID No.3::5 '-AAT TAG GTT CTT GAA GCC CAT CT-3 '；

Sw-5 R is SEQ ID No.4:5 '-TTC CGC ATC AGC CAA TAG TGT-3 '；

(2) CTAB method is used, tomato leaf DNA to be detected is extracted from tomato leaf；

(3) DNA obtained is extracted as template using step (2), utilizeI-2WithSw-5Primer, pass through the multi-PRC reaction of foundation System carries out multiplexed PCR amplification to sample to be tested；

(4) PCR product is subjected to 1.5% agarose gel electrophoresis, is imaged using gel imaging system.

Further, in step (2), specific steps are as follows:

(1) it weighs the tomato leaf of 1g, removes middle arteries, in the mortar after being placed in high-temperature sterilization.

(2) tomato leaf in the mortar in step (1) is ground, when tomato leaf becomes pale green powder shape, rapidly will It is fitted into 5 mL centrifuge tubes.

(3) the CTAB extracting solution of beta -mercaptoethanol and 3 mL that 200 μ L are added into 5 mL centrifuge tubes of step (2) is simultaneously Concussion mixes it uniformly, and wherein CTAB extracting solution is placed in 65 DEG C of water-baths in advance and is preheated.

(4) (3) 5 mL centrifuge tube of step is put into 65 DEG C of 1 h of thermostat water bath, and is shaken once, sufficiently every 10 min It mixes.

(5) be placed in a centrifuge the centrifuge tube of step (4), centrifuge setting: 12000 r/min are centrifuged 10 min；

(6) supernatant solution is taken, is placed in the centrifuge tube of another 5 mL, the supernatant solution volume taken with this step is added Equal phenol: chloroform: isoamyl alcohol, phenol: chloroform: isoamyl alcohol is volume ratio 25:24:1, turns upside down and mixes at least 10 min；

(7) be placed in a centrifuge the centrifuge tube that step (6) obtains, centrifuge setting: 12000 r/min are centrifuged 10 min；

(8) Aspirate supernatant is placed in another 5 mL centrifuge tube, be added chloroform: isoamyl alcohol, the chloroform: isoamyl alcohol it is total Volume is equal with the supernatant volume, and the volume ratio of chloroform and isoamyl alcohol is 24:1, turns upside down and mixes at least 10 min；

(9) be placed in a centrifuge the centrifuge tube of step (8), centrifuge setting: 12000 r/min are centrifuged 10 min；

(10) it takes supernatant solution and is added in another centrifuge tube, the isopropanol of 1.5 times of volumes of supernatant solution is added, mix Even, in -20 DEG C of 30 min of placement, the isopropanol is pre-chilled at -20 DEG C in advance.

(11) centrifuge tube is taken out, is placed in a centrifuge, centrifuge setting: 12000 r/min are centrifuged 10 min.

(12) abandon supernatant, with volume fraction be 70% ethanol washing obtained by white precipitate three times, this precipitating be DNA.

(13) precipitating that is obtained with dehydrated alcohol rinsing step (12) and after drying, is added the TE dissolving DNA of 0.2 mL, RNaseA in the TE containing 0.4 μ L, 10 mg/mL；It is spare that the DNA is diluted to 10-50 ng/ μ L after dissolution.

Further, in step (3), multi-PRC reaction system is 25 μ L solution systems, component and content are as follows: 2 × Template DNA 1.5 the μ L, 10 μm of ol/L of Es Taq MasterMix enzyme 12.5 μ L, 10 ng/ μ LSw-5The upstream and downstream of gene Primer each 0.5 μ L, 10 μm of ol/LI-2Each 1 μ L of the upstream and downstream primer of gene, ultrapure water supply reaction system.

Further, in step (3), multiplexed PCR amplification program are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s；53℃ Anneal 1min；72 DEG C of extension 1min carry out 32 circulations；Last 72 DEG C of extensions 8min.

Further, the product obtained is saved at 4 DEG C.

Further, step (4) is in sample to be testedSw-5WithI-2Carry out identification method specific steps are as follows: in step (4) It is corresponding that 574bp band is found out in the polymorphic bands obtained after electrophoresis detectionSw-5Gene；760bp and 633bp is correspondingI-2Base Cause;464bp is correspondingsw-5Gene；760bp and 693bp is correspondingi-2Gene.If contained only in electrophoresis result 574bp, 760bp and When 633bp band, illustrate in sample to be tested simultaneously containing homozygosisSw-5WithI-2Gene；If being free of 574bp in electrophoresis result Or/and when 760bp and 633bp band, illustrate to be free of in sample to be testedSw-5Or/andI-2Gene.

Further, in step (4), amplified fragments are analyzed,

Genotype isSw-5/Sw-5、I-2/I-2Disease-resistant homozygote material, 760bp, 633bp, 574bp tri- is obtained after expanding Specific band；

Genotype issw-5/sw-5、i-2/i-2Susceptible homozygote material, 760bp, 693bp, 464bp tri- is obtained after expanding Specific band；

Genotype isSw-5/Sw-5、i-2/i-2Material, tri- special items of 760bp, 693bp, 574bp are obtained after expanding Band；

Genotype isSw-5/sw-5、I-2/i-2Material, 760bp, 693bp, 633bp, 574bp, 464bp are obtained after expanding Five specific bands.

Further, in step (4), using 1.5% Ago-Gel, specific step is as follows for electrophoresis:

A) it 1.5% Ago-Gel preparation: weighs agarose and is placed in conical flask, 1 × TAE is added, microwave stove heating is boiled Melt agarose completely, shake up, be cooled to 60 DEG C, 1% red plus nucleic acid dye is added；

B) prepared by offset plate: taking poly (methyl methacrylate) plate and gel maker in electrophoresis tank to clean up, dries, poly (methyl methacrylate) plate level is put Enter in gel maker and fix, be plugged the comb of adaptation, the mixing of Ago-Gel solution is poured on the glass plate in gel device, Glue is unfolded slowly, until entire glass pane surface forms uniform glue-line；It is stood at room temperature until gel solidifies completely, vertically Comb is extracted, gel is put into electrophoresis tank, 1 × TAE electrophoretic buffer is added until not crossing offset plate 1-2mm；

C) it is loaded: sample and sample-loading buffer being mixed, are added in the loading wells of gel；

D) electrophoresis: the gel after sample-adding carries out electrophoresis, voltage 80-100V, electrophoresis 40-60min immediately；

E) observation photograph: being observed using gel imaging system and preservation of taking pictures.

Good effect of the present invention is as follows:

The present invention according to tomato disease-resistant geneSw-5 and I-2Specific primer is designed, foundation can identify two kinds of genes simultaneously Double PCR system, to tomatoSw-5 and I-2Gene carries out double PCR amplification, can efficiently and accurately identify that tomato is disease-resistant miscellaneous Conjunction, homozygous genotype and susceptible genotype.Using the present invention, can save to two kinds of disease-resistant genes of tomato and meanwhile detection time and Cost, testing result can effectively assist tomato breeding for disease-resistance.

Detailed description of the invention

Fig. 1 is the amplification electrophoretogram of comparative example double PCR of the present invention reaction；

Fig. 2 is the amplification electrophoresis that the double PCR of 63 parts of different genotype tomato breeding materials 1-9 of case study on implementation of the present invention reacts Figure；

Fig. 3 is the amplification electricity that the double PCR of 63 parts of different genotype tomato breeding materials 10-18 of case study on implementation of the present invention reacts Swimming figure；

Fig. 4 is the amplification electricity that the double PCR of 63 parts of different genotype tomato breeding materials 19-27 of case study on implementation of the present invention reacts Swimming figure；

Fig. 5 is the amplification electricity that the double PCR of 63 parts of different genotype tomato breeding materials 28-36 of case study on implementation of the present invention reacts Swimming figure；

Fig. 6 is the amplification electricity that the double PCR of 63 parts of different genotype tomato breeding materials 37-45 of case study on implementation of the present invention reacts Swimming figure；

Fig. 7 is the amplification electricity that the double PCR of 63 parts of different genotype tomato breeding materials 46-54 of case study on implementation of the present invention reacts Swimming figure；

Fig. 8 is the amplification electricity that the double PCR of 63 parts of different genotype tomato breeding materials 55-63 of case study on implementation of the present invention reacts Swimming figure.

Specific embodiment

Tomato disease-resistant gene is detected simultaneously the present invention provides a kind ofSw-5WithI-2Multiple PCR technique comprising it is following Step,

One, design of primers

I-2Primer are as follows:

I-2 F is SEQ ID No.1:5 '-CAAGGAACTGCGTCTGTCTG-3 '；

I-2 R is SEQ ID No.2:5 '-ATGAGCAATTTGTGGCCATG-3 '；

According toSw-5Gene order design primer,

Sw-5Primer are as follows:

Sw-5 F is SEQ ID No.3:5 '-AAT TAG GTT CTT GAA GCC CAT CT-3 '；

Sw-5R is SEQ ID No.4:5 '-TTC CGC ATC AGC CAA TAG TGT-3 '；

Two, using CTAB method, tomato leaf DNA to be detected is extracted from tomato leaf；

Further, specific steps are as follows:

(1) tomato leaf of 1g is weighed, it is preferable that be tomato tender leaf, remove middle arteries, the mortar after being placed in high-temperature sterilization In, it is preferable that while the PVP of addition 2% thereto, play the role of removing polyphenols.

(2) the tomato tender leaf in the mortar in step (1) is ground, it is fast when tomato tender leaf becomes pale green powder shape Speed is loaded into 5 mL centrifuge tubes.

Preferably, appropriate liquid nitrogen is added in the mortar in step (1) and is ground rapidly, liquid nitrogen, which plays, makes tomato leaf Piece becomes fragile, and is easy to the effect ground.

(3) beta -mercaptoethanol of 200 μ L and the CTAB extracting solution of 3 mL are added into the centrifuge tube of step (2) and shakes Mix it uniformly, needed for CTAB extracting solution be placed in 65 DEG C of water-baths and preheated in advance.The CTAB is extracted The mass fraction concentration of liquid is 2%.

(4) it takes 5 mL centrifuge tubes of step (3) to be put into 65 DEG C of 1 h of thermostat water bath, and is shaken once, sufficiently every 10 min It mixes.

(5) after the completion of water-bath, the centrifuge tube of step (4) is placed in a centrifuge, centrifuge setting: 12000 r/min, It is centrifuged 10 min；

(6) supernatant solution is taken, is placed in the centrifuge tube of another 5 mL, phenol: chloroform: isoamyl alcohol, phenol: chloroform: isoamyl is added The total volume of alcohol is equal with the supernatant solution volume that this step is taken, phenol: chloroform: isoamyl alcohol is volume ratio 25:24:1, is run up and down Mix at least 10 min.

(7) be placed in a centrifuge the centrifuge tube of step (6), centrifuge setting: 12000 r/min are centrifuged 10 min.

(8) Aspirate supernatant is placed in another 5 mL centrifuge tube, and chloroform: isoamyl alcohol, the chloroform: isoamyl alcohol is added Total volume it is equal with the supernatant volume, chloroform: the volume ratio of isoamyl alcohol be 24:1, turn upside down mix at least 10 min.

(10) it takes supernatant solution and is added in another new centrifuge tube, -20 DEG C of pre-coolings of 1.5 times of volumes of supernatant solution are added Isopropanol, mix, 30 min are placed in -20 DEG C of refrigerator.

(13) dehydrated alcohol rinsing step (12) obtains precipitating and after drying, is added the TE of 0.2 mL, the TE is RNaseA containing 0.4 μ L, 10 mg/mL in Tris-EDTA buffer solution, the TE, makes it dissolve DNA；Dissolution It is spare that the DNA is diluted to 10-50 ng/ μ L with sterile water afterwards.

Three, the DNA obtained is extracted as template using step 2, utilizedI-2WithSw-5Primer, pass through the multiplex PCR of foundation Reaction system carries out multiplexed PCR amplification to sample to be tested；

In step 3, multi-PRC reaction system is 25 μ L solution systems, component and content are as follows: 2 × Es Taq The 1.5 μ L of template DNA of MasterMix enzyme 12.5 μ L, 10 ng/ μ L, the upstream and downstream primer each 0.5 of 10 μm of ol/L Sw-5 genes μ L, each 1 μ L of the upstream and downstream primer of 10 μm of ol/L I-2 genes, ultrapure water supply reaction system to 25 μ L.

In step 3, multiplexed PCR amplification program are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s；53 DEG C of annealing 1min；72 DEG C extend 1min, carry out 32 circulation；Last 72 DEG C of extensions 8min.

Obtained product is saved at 4 DEG C.

Four, PCR product is subjected to 1.5% agarose gel electrophoresis, is imaged using gel imaging system.

Using 1.5% Ago-Gel, specific step is as follows for electrophoresis:

A) it 1.5% Ago-Gel preparation: weighs agarose and is placed in conical flask, 1 × TAE is added, microwave stove heating is boiled Agarose melt completely, shake up, be cooled to 60 DEG C, 1% red plus nucleic acid dye is added；

B) prepared by offset plate: taking poly (methyl methacrylate) plate and gel maker in electrophoresis tank to clean up, dries, poly (methyl methacrylate) plate level is put Enter in gel maker and fix, be plugged suitable comb, Ago-Gel solution is mixed to the glass carefully poured into gel device On plate, glue is unfolded slowly, until entire glass pane surface forms uniform glue-line；It is stood at room temperature until gel is completely solidifying Gu vertically gently extracting comb, gel is put into electrophoresis tank, 1 × TAE electrophoretic buffer is added until not crossing offset plate 1-2mm；

In step (4), in sample to be testedSw-5WithI-2Carry out identification method specific steps are as follows: examine in step 4 electrophoresis The 574bp band found out in the polymorphic bands obtained after survey is correspondingSw-5Gene；760bp and 633bp is correspondingI-2Gene； 464bp is correspondingsw-5Gene；760bp and 693bp is correspondingi-2Gene.If contained only in electrophoresis result 574bp, 760bp and When 633bp band, illustrate in sample to be tested simultaneously containing homozygosisSw-5WithI-2Gene；If being free of 574bp in electrophoresis result Or/and when 760bp and 633bp band, illustrate in sample to be tested without disease-resistant geneSw-5Or/andI-2Gene.

Amplified fragments are analyzed,

It is that genotype is that tri- specific bands of 760bp, 633bp, 574bp are obtained after expandingSw-5/Sw-5、I-2/I-2's Disease-resistant homozygote material；

It is that genotype is that tri- specific bands of 760bp, 693bp, 464bp are obtained after expandingsw-5/sw-5、i-2/i-2Sense Sick homozygote material；

It is that genotype is that tri- specific bands of 760bp, 693bp, 574bp are obtained after expandingSw-5/Sw-5、i-2/i-2Material Material；

It is that genotype is that five specific bands of 760bp, 693bp, 633bp, 574bp, 464bp are obtained after expandingSw-5/sw-5、I-2/i-2Material.

Illustrate,Sw-5/ Sw-5For with anti-tomato spotted wilf virus ospc geneSw-5Disease-resistant homozygote；

Sw-5/ sw-5For with anti-tomato spotted wilf virus ospc geneSw-5Disease-resistant heterozygote；

sw-5/ sw-5For without anti-tomato spotted wilf virus ospc geneSw-5Susceptible homozygote.

I-2/ I-2For with anti-tomato wilt geneI-2Disease-resistant homozygote；

I-2/ i-2For with anti-tomato wilt geneI-2Disease-resistant heterozygote；

i-2/i-2For without anti-tomato wilt geneI-2Susceptible homozygote.

Embodiment

Detailed description below detection process of the invention:

1 test material

Choose the 7 Tomato Breeding Lines of unknown gene type totally 63 parts be experimental material, the concrete condition of 63 parts of experimental materials is shown in Table 1- table 2.The sowing of tomato material is subjected to nursery on porous disc, seedling is numbered one by one when growing 4 to 5 pieces of true leaves, and from Top spire 2 to 3 is picked on every single plant, and it is spare to extract spire genomic DNA.

1 63 parts of tomato breeding material 1-32 of table

2 63 parts of tomato breeding material 33-63 of table

2. test method:

Test material is identified using the duplex PCR reaction of acquisition.Pcr amplification reaction is carried out to genomic DNA.PCR body System is same as above.Amplified production carries out electrophoresis detection with 1.5% Ago-Gel, finally observes result with gel imaging system.

Specific steps are as follows:

One, design of primers

I-2Primer are as follows:

I-2 F is SEQ ID No.1:5 '-CAAGGAACTGCGTCTGTCTG-3 '；

I-2 R is SEQ ID No.2:5 '-ATGAGCAATTTGTGGCCATG-3 '；

According toSw-5Gene order design primer,

Sw-5Primer are as follows:

Sw-5F is SEQ ID No.3:5 '-AAT TAG GTT CTT GAA GCC CAT CT-3 '；

Sw-5R is SEQ ID No.4:5 '-TTC CGC ATC AGC CAA TAG TGT-3 '；

Further, specific steps are as follows:

(1) from 63 parts of tomato leaf samples, spire is chosen, weighs 1g tomato leaf respectively, middle arteries is removed, is placed in high temperature In mortar after sterilizing, it is preferable that while the PVP of addition 2% thereto, play the role of removing polyphenols.

(2) appropriate liquid nitrogen is added in the mortar in step (1) and the tomato tender leaf in mortar is ground rapidly, to tomato When tender leaf becomes pale green powder shape, it is loaded into rapidly in 5 mL centrifuge tubes.

(3) beta -mercaptoethanol and 3 of 200 μ L is added in the centrifuge tube for accommodating pale green powder shape product to step (2) The CTAB extracting solution of mL and shaking mixes it uniformly, needed for CTAB extracting solution be placed in 65 DEG C of water-baths in advance into Row preheating.The CTAB extracting solution mass fraction concentration is 2%.

(5) after the completion of water-bath, the centrifuge tube that step (4) obtains is placed in a centrifuge, centrifuge setting: 12000 r/ Min is centrifuged 10 min；

(6) supernatant solution is taken, is placed in the centrifuge tube of another 5 mL, phenol: chloroform: isoamyl alcohol, phenol: chloroform: isoamyl is added Alcohol addition volume is equal with the supernatant solution volume that this step is taken, phenol: chloroform: isoamyl alcohol is volume ratio 25:24:1, is run up and down Mix at least 10 min.

(7) centrifuge tube that step (6) obtains is placed in a centrifuge, centrifuge setting: 12000 r/min, centrifugation 10 min。

(10) take supernatant solution and be added in another new centrifuge tube, be added 1.5 volume of supernatant solution at -20 DEG C Pre- cold isopropanol mixes, 30 min is placed in -20 DEG C of refrigerator.

(12) supernatant is abandoned, three times, this precipitating is DNA to the white precipitate obtained by 70% ethanol washing.

(13) after dehydrated alcohol is rinsed and dried, the TE of 0.2 mL is added, the TE is Tris-EDTA buffer RNaseA containing 0.4 μ L, 10 mg/mL in solution, the TE, makes it dissolve DNA；It will be described with sterile water after dissolution It is spare that DNA is diluted to 10-50 ng/ μ L.

In step 3, multi-PRC reaction system is 25 μ L solution systems, component and content are as follows: 2 × Es Taq Template DNA 1.5 the μ L, 10 μm of ol/L of MasterMix enzyme 12.5 μ L, 10 ng/ μ LSw-5The upstream and downstream primer of gene each 0.5 μ L, 10 μm of ol/LI-2Each 1 μ L of the upstream and downstream primer of gene, ultrapure water supply reaction system to 25 μ L.

Obtained product is saved at 4 DEG C.

Using 1.5% Ago-Gel, specific step is as follows for electrophoresis:

As a result as illustrated in figs. 2 through 8, note: M:100 bp Ladder, 1-63: the number of different genotype breeding material；

Amplified fragments are analyzed,

The genotype that tri- specific bands of 760bp, 633bp, 574bp are obtained after amplification isSw-5/Sw-5、I-2/I-2It is anti- Sick homozygote material,

The genotype that tri- specific bands of 760bp, 693bp, 464bp are obtained after amplification issw-5/sw-5、i-2/i-2It is susceptible Homozygote material,

The genotype that tri- specific bands of 760bp, 693bp, 574bp are obtained after amplification isSw-5/Sw-5、i-2/i-2It is disease-resistant Material,

The genotype that five specific bands of 760bp, 693bp, 633bp, 574bp, 464bp are obtained after amplification isSw-5/sw-5、I- 2/i-2Disease-resistant material,

Testing result:

63 parts of breeding materials are detected using the double PCR system that the present invention obtains, acquired results are as illustrated in figs. 2 through 8.? In 63 parts of test materials, containSw-5/Sw-5 WithI-2/I-21 part of disease-resistant homozygote material of gene, amplify 760 bp, 633 bp, 574 bp, tri- specific bands.Containsw-5/sw-5Withi-2/i-223 parts of susceptible homozygote material of gene expand Increase 760 bp, 693 bp, 464 bp, tri- specific bands out.Contain sw-5/sw-5、I-2/I-2The disease-resistant material 25 of gene Part, tri- specific bands of 760bp, 633bp, 464bp are obtained after expanding；ContainSw-5/sw-5、I-2/i-2Gene it is disease-resistant 3 parts of material, five specific bands of 760bp, 693bp, 633bp, 574bp, 464bp are obtained after expanding.ContainSw-5/sw-5、 I-2/I-21 part of the disease-resistant material of gene obtains tetra- specific bands of 760bp, 633bp, 574bp and 464bp after expanding.Contain HaveSw-5/sw-5、i-2/i-25 parts of the material of gene obtains tetra- special items of 760bp, 693bp, 574bp and 464bp through amplification Band.

The genotype of tomato breeding material can be detected using the double PCR system, while filtering out and containing simultaneouslySw-5/ Sw-5WithI-2/I-2Compound 1 part of the disease-resistant homozygote material of genotype.

Comparative example

With anti-tomato spotted wilf virus ospc geneSw-5Disease-resistant homozygote (Sw-5/ Sw-5), disease-resistant heterozygote (Sw-5/ sw- 5), susceptible homozygote (sw-5/ sw-5), anti-tomato wilt geneI-2Disease-resistant homozygote (I-2/ I-2), disease-resistant heterozygote (I-2/ i-2), susceptible homozygote (i-2/i-2) breeding material, sowing nursery is carried out on porous disc, 3-4 piece to be grown is true Ye Shi takes the young leaflet tablet of upper end as test material.

Using same embodiment same operation step, only the tomato spire of experimental subjects is different, other operations are identical, By step 1 to four, the double PCR system of acquisition detects above-mentioned breeding material, using gel imaging system at Picture, acquired results are as shown in Figure 1.

Genotype isSw-5/Sw-5、I-2/I-2Disease-resistant homozygote material, obtained after expanding 760bp, 633bp, Tri- specific bands of 574bp；

Genotype isSw-5/sw-5、I-2/I-2Material, 760bp, 633bp, 574bp and 464bp tetra- are obtained after expanding Specific band.

Genotype isSw-5/sw-5、i-2/i-2Material, through amplification obtain 760bp, 693bp, 574bp and 464bp tetra- Specific band.

Genotype isSw-5/sw-5、I-2/i-2Material, obtained after expanding 760bp, 693bp, 633bp, 574bp, Five specific bands of 464bp.

Heretofore described tomatoSw-5Gene andI-2The multi-PCR detection method of gene can detect simultaneouslySw-5Gene WithI-2Two kinds of disease-resistant genes of gene, accelerate the process to molecular mark, shorten breeding time, reduce and educate Kind cost.

The present invention according to tomato disease-resistant geneSw-5 and I-2Specific primer is designed, foundation can identify two kinds simultaneously The double PCR system of gene, to tomatoSw-5 and I-2Gene carries out double PCR amplification, can efficiently and accurately identify that tomato is anti- Sick heterozygosis, homozygous genotype and susceptible genotype.Using the present invention, can save to two kinds of disease-resistant genes of tomato and meanwhile detection when Between and cost, testing result can effectively assist tomato breeding for disease-resistance.

The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one The widest scope of cause.

SEQUENCE LISTING

<110>Hebei University of Science and Technology

<120>a kind of multiple PCR method for detecting tomato disease-resistant gene Sw-5 and I-2 simultaneously

<130> 2018

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213>artificial sequence

<400> 1

caaggaactg cgtctgtctg 20

<210> 2

<211> 20

<212> DNA

<213>artificial sequence

<400> 2

atgagcaatt tgtggccatg 20

<210> 3

<211> 23

<212> DNA

<213>artificial sequence

<400> 3

aattaggttc ttgaagccca tct 23

<210> 4

<211> 21

<212> DNA

<213>artificial sequence

<400> 4

ttccgcatca gccaatagtg t 21

Claims

1. a kind of detect tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR method, it is characterised in that: it includes following step Suddenly,

(1) design of primers,

I-2Primer are as follows:

I-2 F:5 '-CAAGGAACTGCGTCTGTCTG-3 '；

I-2 R:5 '-ATGAGCAATTTGTGGCCATG-3 '；

Sw-5Primer are as follows:

Sw-5 F:5 '-AAT TAG GTT CTT GAA GCC CAT CT-3 '；

Sw-5 R:5 '-TTC CGC ATC AGC CAA TAG TGT-3 '；

2. one kind according to claim 1 detects tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR method, it is special Sign is: in step (2), specific steps are as follows:

(1) it weighs the tomato leaf of 1g, removes middle arteries, in the mortar after being placed in high-temperature sterilization;

(2) tomato leaf in the mortar in step (1) is ground, when tomato leaf becomes pale green powder shape, is filled rapidly Enter in 5 mL centrifuge tubes;

(3) beta -mercaptoethanol of 200 μ L and the CTAB extracting solution of 3 mL are added into 5 mL centrifuge tubes of step (2) and shakes Mix it uniformly, wherein CTAB extracting solution is placed in 65 DEG C of water-baths in advance and is preheated;

(4) (3) 5 mL centrifuge tube of step is put into 65 DEG C of 1 h of thermostat water bath, and is shaken once every 10 min, mixed well;

(10) it takes supernatant solution and is added in another centrifuge tube, the isopropanol of 1.5 times of volumes of supernatant solution is added, mix Even, in -20 DEG C of 30 min of placement, the isopropanol is pre-chilled at -20 DEG C in advance;

(11) centrifuge tube is taken out, is placed in a centrifuge, centrifuge setting: 12000 r/min are centrifuged 10 min;

(12) abandon supernatant, with volume fraction be 70% ethanol washing obtained by white precipitate three times, this precipitating be DNA;

(13) precipitating that is obtained with dehydrated alcohol rinsing step (12) and after drying, is added the TE dissolving DNA of 0.2 mL, described RNaseA in TE containing 0.4 μ L, 10 mg/mL；It is spare that the DNA is diluted to 10-50 ng/ μ L after dissolution.

3. one kind according to claim 1 detects tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR method, it is special Sign is: in step (3), multi-PRC reaction system is 25 μ L solution systems, component and content are as follows: 2 × Es Taq Template DNA 1.5 the μ L, 10 μm of ol/L of MasterMix enzyme 12.5 μ L, 10 ng/ μ LSw-5The upstream and downstream primer of gene is each 0.5 μ L, 10 μm of ol/LI-2Each 1 μ L of the upstream and downstream primer of gene, ultrapure water supply reaction system.

4. one kind according to claim 1 detects tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR method, it is special Sign is: in step (3), multiplexed PCR amplification program are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s；53 DEG C of annealing 1min；72 DEG C extend 1min, carry out 32 circulation；Last 72 DEG C of extensions 8min.

5. one kind according to claim 4 detects tomato disease-resistant gene simultaneouslySw-5 HesI-2Multiple PCR method, it is special Sign is: obtained product is saved at 4 DEG C.

6. one kind according to claim 1 detects tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR method, it is special Sign is: in step (4), amplified fragments analyzed,

7. one kind according to claim 1 detects tomato disease-resistant gene simultaneouslySw-5WithI-2Multiple PCR method, it is special Sign is: in step (4), using 1.5% Ago-Gel, specific step is as follows for electrophoresis: