CN105296475A - Molecular marker interlocked with pepper cucumber mosaic virus disease resistance gene qcmv-2-1 and application of molecular marker - Google Patents
Molecular marker interlocked with pepper cucumber mosaic virus disease resistance gene qcmv-2-1 and application of molecular marker Download PDFInfo
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Abstract
The invention discloses a molecular marker interlocked with a pepper cucumber mosaic virus disease resistance gene qcmv-2-1 and an application of the molecular marker. According to the molecular marker interlocked with the pepper cucumber mosaic virus disease resistance gene qcmv-2-1, pepper genome DNA is amplified through a primer pair InDel-2-134F/InDel-2-134R, and an obtained 230bp amplified fragment is the molecular marker interlocked with the pepper cucumber mosaic virus disease resistance gene qcmv-2-1; the InDel-2-134F sequence is shown as SEQ ID NO.1, and the InDel-2-134R sequence is shown as SEQ ID NO.2. According to the Indel molecular marker and corresponding primers interlocked with the qcmv-2-1 resistance gene and screened through the InDel marker and QTL-Seq technology, marker bands are simple, and the molecular marker and the application of the molecular marker can be directly used for marker-assisted section.
Description
Technical field
The invention belongs to field of molecular breeding, relate to the molecule marker chain with capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1 and application thereof.
Background technology
Cucumber mosaic virus (Cucumbermosaicvirus, CMV) be the prototypical member of Bromoviridae (Bromoviridae) Cucumovirus (Cucumovirus), the systemic symptom that capsicum is serious can be caused, cause yellowing leaf, torsional deformation, serious floral leaf, the commodity property of infringement fruit.Cucumber Mosaic Virus generally can cause the capsicum underproduction 20% ~ 30%, can reach 50% ~ 60% time serious, and indivedual area even has no harvest, and is one of the most serious disease of capsicum.During China capsicum produces, CMV recall rate is the highest, is the main goal of attack of pepper disease resistance breeding.
In traditional breeding way, the disease-resistant material of seed selection is by inoculated identification, and the phenotype according to plant is screened.This kind of method, because inoculation is insufficient or onset condition is not suitable for and affects efficiency of selection, is difficult to filter out accurately and rapidly the individual plant with disease-resistant gene.In addition capsicum cucumber-mosaic-virus resistant ospc gene major part is from Wild pepper, and disease-resistant gene is normal with bad character gene close linkage.Bad chain required colony is large, the cycle is long, efficiency is low to utilize traditional breeding way to break, and molecular marker assisted selection (MolecularMarker-assistedSelection, MAS) breeding effectively can overcome the defect of traditional breeding method, accelerates breeding process.
Insertion/deletion (insertion/deletion, InDel) is because the DNA sequence dna of allelotrope site there occurs the insertion/deletion of nucleotide fragments and the length polymorphism variation that produces between Different Individual.Sequences Design special primer according to target site both sides carries out pcr amplification, and namely the length polymorphism of amplified fragments is InDel mark.In whole genome, the frequency of polymorphism of Indel mark is only second to SNP marker, far above SSR marker.InDel marks polymorphism reaches gene type object by polymerase chain reaction (PCR) and the simple step such as agarose gel electrophoresis or native polyacrylamide gel electrophoresis.InDel is labeled as codominant marker, has good stability and the polymorphism compared with horn of plenty, is widely used in the fields such as high resolving power collection of illustrative plates structure, association analysis, the assignment of genes gene mapping and objective trait molecular marker screening.The present invention adopts QTL-Seq method and Indel labeling technique, screening and the closely linked mark of Cucumber Mosaic Virus resistant gene qcmv-2-1, to realize the molecular mark of Cucumber Mosaic Virus resistance, accelerate the seed selection process of the sick new variety of China's capsicum cucumber-mosaic-virus resistant.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of molecule marker chain with capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1 is provided.
Another object of the present invention is to provide the primer pair of the molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1.
Another object of the present invention is to provide the application of this molecule marker and primer pair thereof.
Object of the present invention realizes by following technical scheme:
A kind of molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1, by primer pair InDel-2-134F/InDel-2-134R amplification capsicum genomic dna, the amplified fragments of the 230bp of acquisition is the described molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1; Wherein, InDel-2-134F sequence is as shown in SEQIDNO.1, and InDel-2-134R sequence is as shown in SEQIDNO.2.
The primer pair InDel-2-134F/InDel-2-134R of the molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1, InDel-2-134F sequence is as shown in SEQIDNO.1, and InDel-2-134R sequence is as shown in SEQIDNO.2.
The molecule marking method of capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1, to increase capsicum genomic dna to be checked with primer pair InDel-2-134F/InDel-2-134RPCR of the present invention, and detect amplified production, if amplify the amplified fragments of 230bp, then indicate in capsicum to be checked to there is capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1, capsicum to be checked is Cucumber Mosaic Virus disease-resistant plant.
The application of molecule marker of the present invention in qualification hot pepper germ plasm resource in Cucumber Mosaic Virus resistant gene qcmv-2-1.
The application of molecule marker of the present invention in the capsicum of screening cucumber-mosaic-virus resistant disease.
The application of molecule marker of the present invention in the molecular breeding cultivating the sick capsicum of cucumber-mosaic-virus resistant.
The application of primer pair InDel-2-134F/InDel-2-134R of the present invention in qualification hot pepper germ plasm resource in Cucumber Mosaic Virus resistant gene qcmv-2-1.
The application of primer pair InDel-2-134F/InDel-2-134R of the present invention in the sick capsicum of screening cucumber-mosaic-virus resistant.
The application of primer pair InDel-2-134F/InDel-2-134R of the present invention in the molecular breeding cultivating the sick capsicum of cucumber-mosaic-virus resistant.
A kind of method of screening the sick capsicum of cucumber-mosaic-virus resistant, to increase capsicum genomic dna to be checked with described primer pair InDel-2-134F/InDel-2-134RPCR, and detect amplified production, if the amplified fragments of 230bp can be amplified, then indicate that capsicum to be checked is the pepper plant that there is capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1.
Beneficial effect:
Cucumber Mosaic Virus is one of virus disease that China's capsicum recall rate is the highest, is the main goal of attack of China's pepper disease resistance breeding.
Invention herein utilizes Indel to mark and QTL-Seq method screens the Indel molecule marker chain with qcmv-2-1 resistant gene and corresponding primer that obtain, and marker bands is simple, can be directly used in marker assisted selection.
This mark is the codominant marker of a PCR-based technology, compared with marking, can significantly reduce costs with RFLP, CAPS etc., saves labour.This tag application, in nursery selection, not only can reduce workload, and can avoid insufficiently being difficult to filter out the individual plant with disease-resistant gene because inoculating, thus accelerates breeding process.
Accompanying drawing explanation
Fig. 1 InDel marks the amplification situation of InDel-2-134 between parent and between anti-sense pond
M:100bpMarker1; P1: disease-resistant parent PBC688; P2: Susceptible parent G29; The disease-resistant individual plant of 1-5:F2; The susceptible individual plant of 6-10:F2
Fig. 2 InDel marks InDel-2-134 at F
2checking in colony
M:100bpmarker; 1-10:F
2for disease-resistant individual plant; 11-20:F
2for susceptible individual plant
Fig. 3 Indel is marked at the checking in 23 parts of anti-sense individual plants of F2
M:100bpmarker; 1-20:20 part F
2disease-resistant individual plant; 21-23:3 part F
2susceptible individual plant
Embodiment
Embodiment 1
1. for examination material
Apply disease-resistant self-mating system PBC688 and (utilize QTL-Seq technological orientation capsicum cucumber-mosaic-virus resistant ospc gene for maternal, gardening journal, 2015, doi:10.16420/j.issn.0513-353x), susceptible system G29 is that the combination of male parent preparing hybrid (utilizes QTL-Seq technological orientation capsicum cucumber-mosaic-virus resistant ospc gene, gardening journal, 2015, doi:10.16420/j.issn.0513-353x), F is obtained
1after, selfing obtains F again
2segregating population, utilizes artificial frictional inoculation cucumber mosaic virus Fny strain inoculated identification in seedling stage (the sick progress of capsicum cucumber-mosaic-virus resistant.North China agronomy report, 2014,29 (supplementary issue): 77-84).
Specific practice is: select full planting seed in nutrition pot, and every alms bowl 1 seed, when 4 leaf 1 heart, carries out artificial frictional inoculation.Inoculate 4 weeks " Invest, Then Investigate " colony incidences, Disease investigation be classified as 0 grade-without any symptom; The bright arteries and veins of 1 grade-lobus cardiacus or the acute little withered spot of inoculation leaf; 3 grades-system floral leaf or stem produce necrotic spot; The heavy floral leaf of 5 grades-system, deformity or stem produce downright bad streak; 7 grades-most leaf malformation, fern leaf, plant downgrade or stem, branch and vein system downright bad; 9 grades-plant is seriously downgraded, stop growing or serious systemic downright bad, to complete stool death (see reference document: Crops In China and wild kindred plant thereof: vegetable crop volume [M]. Beijing: Chinese agriculture press, 2008:719-720).
2. the extraction of genomic dna
Pepper Leaves puts into 1.5ml centrifuge tube, and liquid nitrogen flash freezer is ground to powder, adds the CTAB extraction buffer that 700 μ l are preheated to 65 DEG C 2% immediately, extracts 30min in 65 DEG C of water-baths, be inverted sample tube gently 3 times during this.Add 700 μ l chloroforms: primary isoamyl alcohol=24:1 extract, mix extracting 5min gently, centrifugal 5 minutes of 12000r/min.Draw 400 μ l supernatant liquors to be transferred in new centrifuge tube, add the dehydrated alcohol of 800 μ l precoolings, shake up gently, the centrifugal 5min of 12000r/min.Outwell dehydrated alcohol, note white DNA pellet not being poured out, add 800 μ l75% ethanol purge 2 times, outwell the alcohol of 75%, precipitate is placed in centrifuge tube bottom, dries under test tube being placed in room temperature, add the sterilizing deionized water (ddH that 100 μ l include RNAseA
2o:RNaseA=50:1), 37 DEG C of water-bath 1h, make DNA dissolve and remove RNA, are placed in-20 DEG C of preservations, for subsequent use.
The analysis of 3.InDel molecule marker
A. anti-sense gene pool builds
Adopt BSA method to build disease-resistant and susceptible gene pool respectively, extract F
2after individual plant DNA, choose extremely disease-resistant and susceptible each 5 individual plant DNA balanced mix.The anti-CMV gene of capsicum is located for QTL-Seq.
B.InDel polymorphism primer screens
Utilize QTL-Seq technology by the qcmv-2-1 assignment of genes gene mapping at No. 2 karyomit(e)s, associated region is 4.26Mb.Develop 30 pairs of Indel primer sequences according to Parent order sequenced data of resurveying, entrust the synthesis of prompt base (Shanghai) trade Co., Ltd in the English Weihe River, Shanghai.The reaction system of InDel mark is: the DNA profiling 0.5 μ l of 50ng/ μ l; The primer pair 0.5 μ l of 10 μm of ol/L; 10 × PCRbuffer1.0 μ l; 25mmol/LMgCl
21.0 μ l; 10mmol/LdNTPs0.25 μ l; 5U/ μ lTaqDNAPolymerase0.1 μ l; ddH
2o6.65 μ l; Or adopt the DNA profiling 0.5 μ l of 50ng/ μ l; The primer pair 0.5 μ l of 10 μm of ol/L; 2 ×
greenMasterMix5 μ l; ddH
2o3 μ l.PCR response procedures is: 94 DEG C of denaturation 4min; Each circulation 94 DEG C of sex change 30sec, 57 DEG C of annealing 30sec, 72 DEG C extend 40sec, totally 30 circulations; Last 72 DEG C extend 7min.PCR primer carries out isolation identification on the non-denaturing polyacrylamide gel of 10%, and the colour developing of silver dye, then observes, records experimental result on visible drop instrument.
B. results and analysis
Utilize above-mentioned 30 pairs of InDel primers to carry out polymorphism screening between resistance parent PBC688 and Susceptible parent G29, result has 16 pairs of primers can amplify polymorphism between anti-sense parent, by polymorphism primer to disease-resistant/the susceptible and F built after inoculation
2colony carries out amplification qualification further, and the polymorphism of amplification is stablized.Molecular marker screening result shows, InDel marks InDel-2-134 and capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1 close linkage, can amplify the fragment of 230bp.
The primer of this mark is:
InDel-2-134F：TGCTTCAGTTGAGTTGTCCA(SEQIDNO.1)
InDel-2-134R：TAAATCCCCTTGTGGTGGCT(SEQIDNO.2)
Namely with the breeding material DNA of primer I nDel-2-134F/InDel-2-134R amplification containing Cucumber Mosaic Virus gene qcmv-2-1, if the fragment of 230bp can be amplified, then the existence of qcmv-2-1 gene is indicated.By to containing the disease-resistant parent PBC688 of qcmv-2-1, Susceptible parent G29, the F that hybridization and selfing obtain
2linkage analysis (analysis software MapQTL4.0) is carried out, Indel-2-134 and capsicum Cucumber Mosaic Virus gene qcmv-2-1 close linkage, linkage distance 0.3cM for 190 individual plant investigation results and molecule marker spectrum data.
In order to detect the reliability of this mark, this experimental applications 20 parts of disease-resistant F
2individual plant and 3 parts of susceptible F
2individual plant (F used
2individual plant passes through F
2:3the anti-CMV qualification of family, qualification result is in table 1) verify, result is as shown in Figure 3,3 parts of susceptible individual plants have amplified the band of 211bp, 7 parts of bands only amplifying 230bp are had, 11 parts of bands that simultaneously can amplify 211bp and 230bp, 2 parts of (F in 20 parts of disease-resistant individual plants
2-54 and F
2-214) band of 211bp has been amplified.These results suggest that 21 strain F
2individual plant marker genetype conforms to Resistance Identification result, 2 strain F
2individual plant (F
2-54 and F
2-214) marker genetype and Resistance Identification result are not inconsistent, so the accuracy rate detected is 91.3%.According to these results suggest that this tag application in molecular marker assisted selection breeding be feasible.
Above-mentioned embodiment is used for explaining and the present invention is described, instead of limits the invention, and in the protection domain required by the moral rights of inventing, any amendment make the present invention and change, all fall into protection scope of the present invention.
23 parts of F that table 1 verification mark reliability is used
2individual plant capsicum material
Note: qcmv-2-1 is anti-CMV gene, and Qcmv-2-1 is the allelotrope of qcmv-2-1.
Claims (10)
1. a molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1, it is characterized in that the 230bp amplified fragments of acquisition is the molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1 by primer pair InDel-2-134F/InDel-2-134R amplification capsicum genomic dna; Wherein, InDel-2-134F sequence is as shown in SEQIDNO.1, and InDel-2-134R sequence is as shown in SEQIDNO.2.
2. the primer pair InDel-2-134F/InDel-2-134R of the molecule marker chain with capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1, it is characterized in that InDel-2-134F sequence is as shown in SEQIDNO.1, InDel-2-134R sequence is as shown in SEQIDNO.2.
3. the molecule marking method of capsicum Cucumber Mosaic Virus resistant gene qcmv-2-1, it is characterized in that increasing capsicum genomic dna to be checked with primer pair InDel-2-134F/InDel-2-134RPCR according to claim 2, and detect amplified production, if amplify the amplified fragments of 230bp, then indicate in capsicum to be checked to there is capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1, capsicum to be checked is Cucumber Mosaic Virus disease-resistant plant.
4. the application of molecule marker according to claim 1 in qualification hot pepper germ plasm resource in Cucumber Mosaic Virus resistant gene qcmv-2-1.
5. the application of molecule marker according to claim 1 in the capsicum of screening cucumber-mosaic-virus resistant disease.
6. the application of molecule marker according to claim 1 in the molecular breeding cultivating the sick capsicum of cucumber-mosaic-virus resistant.
7. the application of primer pair InDel-2-134F/InDel-2-134R according to claim 2 in qualification hot pepper germ plasm resource in Cucumber Mosaic Virus resistant gene qcmv-2-1.
8. the application of primer pair InDel-2-134F/InDel-2-134R according to claim 2 in the sick capsicum of screening cucumber-mosaic-virus resistant.
9. the application of primer pair InDel-2-134F/InDel-2-134R according to claim 2 in the molecular breeding cultivating the sick capsicum of cucumber-mosaic-virus resistant.
10. one kind is screened the method for the sick capsicum of cucumber-mosaic-virus resistant, it is characterized in that increasing capsicum genomic dna to be checked with primer pair InDel-2-134F/InDel-2-134RPCR according to claim 2, and detect amplified production, if the amplified fragments of 230bp can be amplified, then indicate that capsicum to be checked is the capsicum that there is capsicum cucumber-mosaic-virus resistant ospc gene qcmv-2-1.
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Cited By (10)
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| CN105907755A (en) * | 2016-06-10 | 2016-08-31 | 中国农业科学院蔬菜花卉研究所 | Molecular marker closely linked with resistance of hot peppers to cucumber mosaic virus and application of molecular marker |
| CN107674922A (en) * | 2017-09-01 | 2018-02-09 | 中国农业科学院蔬菜花卉研究所 | Cucumber anti cucumber mosaic virus ospc gene cmv InDel marks and its application |
| CN108048601A (en) * | 2017-12-20 | 2018-05-18 | 江苏省农业科学院 | A kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application |
| CN108315465A (en) * | 2018-03-27 | 2018-07-24 | 江苏省农业科学院 | A kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings and its primer and application |
| CN108517373A (en) * | 2018-05-23 | 2018-09-11 | 江苏省农业科学院 | It one InDel labeled primer pair for distinguishing five pepper cultivation kinds and its applies |
| CN108950054A (en) * | 2018-08-28 | 2018-12-07 | 江苏省农业科学院 | A kind of InDel molecular marker and primer thereof and application with cowpea salt tolerant correlated traits close linkage |
| CN109913532A (en) * | 2019-04-11 | 2019-06-21 | 江苏省农业科学院 | A method of obtaining sponge gourd anti cucumber mosaic virus disease candidate gene |
| CN111163630A (en) * | 2017-09-29 | 2020-05-15 | 塞米尼斯蔬菜种子公司 | Cucumber mosaic virus resistant pepper plants |
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| CN105907755A (en) * | 2016-06-10 | 2016-08-31 | 中国农业科学院蔬菜花卉研究所 | Molecular marker closely linked with resistance of hot peppers to cucumber mosaic virus and application of molecular marker |
| CN107674922A (en) * | 2017-09-01 | 2018-02-09 | 中国农业科学院蔬菜花卉研究所 | Cucumber anti cucumber mosaic virus ospc gene cmv InDel marks and its application |
| CN111163630A (en) * | 2017-09-29 | 2020-05-15 | 塞米尼斯蔬菜种子公司 | Cucumber mosaic virus resistant pepper plants |
| CN108048601A (en) * | 2017-12-20 | 2018-05-18 | 江苏省农业科学院 | A kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application |
| CN108048601B (en) * | 2017-12-20 | 2021-06-08 | 江苏省农业科学院 | Seedling inoculation method for pepper cucumber mosaic virus and application thereof |
| CN108315465A (en) * | 2018-03-27 | 2018-07-24 | 江苏省农业科学院 | A kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings and its primer and application |
| CN108315465B (en) * | 2018-03-27 | 2021-07-06 | 江苏省农业科学院 | InDel molecular marker closely linked with cowpea salt tolerance related characters, primers and application thereof |
| CN108517373A (en) * | 2018-05-23 | 2018-09-11 | 江苏省农业科学院 | It one InDel labeled primer pair for distinguishing five pepper cultivation kinds and its applies |
| CN108517373B (en) * | 2018-05-23 | 2021-10-19 | 江苏省农业科学院 | InDel labeled primer pair for distinguishing five pepper cultivars and application thereof |
| CN108950054A (en) * | 2018-08-28 | 2018-12-07 | 江苏省农业科学院 | A kind of InDel molecular marker and primer thereof and application with cowpea salt tolerant correlated traits close linkage |
| CN109913532A (en) * | 2019-04-11 | 2019-06-21 | 江苏省农业科学院 | A method of obtaining sponge gourd anti cucumber mosaic virus disease candidate gene |
| CN111903448A (en) * | 2020-08-24 | 2020-11-10 | 云南省农业科学院生物技术与种质资源研究所 | Chili plant cultivation method |
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