CN100447254C - Molecular marker-assisted selection method for capsicum blight-resistant breeding - Google Patents
Molecular marker-assisted selection method for capsicum blight-resistant breeding Download PDFInfo
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- CN100447254C CN100447254C CNB200610123860XA CN200610123860A CN100447254C CN 100447254 C CN100447254 C CN 100447254C CN B200610123860X A CNB200610123860X A CN B200610123860XA CN 200610123860 A CN200610123860 A CN 200610123860A CN 100447254 C CN100447254 C CN 100447254C
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Abstract
The invention discloses a molecular marked auxiliary selecting method of pepper pestilence-proof breeding, which is characterized by the following: sowing pepper pestilence-proof breeding separated generation material into greenhouse; fetching 1-2 spire from each plant to extract DNA after the spire grew 5-6h; adopting DNA as mold; utilizing SCAR marked two-primer to proceed PCR augumentation; annealing under 56-58 deg.c; separating augumented product on the 1.5% agarose gel electrophoresis; dyeing through ethidium bromide; observing PCR result; augumenting DNA band with 520bp.
Description
Technical field
The invention belongs to the field of crop breeding and crop molecular marker breeding technology, particularly a kind of molecular marker-assisted selection method of capsicum blight-resistant breeding.
Background technology
Capsicum epidemic disease (Phytophthora capsici L) is that capsicum produces one of severe diseases in the world wide.Capsicum epidemic disease has brone infection and two kinds of character of soil infection, but based on the soil propagation, and morbidity is sudden strong in addition, and epidemic rate is exceedingly fast, thereby chemical control and cultural control measure effect are not good.Cultivating antilemic capsicum variety is to address this problem most economical valid approach.Filter out some antilemic capsicum resources with the artificial inoculation authenticate technology both at home and abroad, but the economical character of these resistance resources and quality trait are relatively poor, can not directly use as breeding material, need be to commercial variety with its good resistance transformation.Traditional resistance transfer method mainly is hybridization, introduces bad economical character and quality trait inevitably like this when importing disease-resistant gene, is badly in need of setting up a kind of more efficiently breeding system of selection in the breeding practice.The setting-up and development of marker assisted selection technology provide effective way for addressing this problem.
Studies show that of lot of domestic and foreign, the heredity of capsicum blight-resistant presents the characteristics of " genetic diversity ", that is: and the resistance of some materials is controlled by single-gene, and some are oligogene control, and other then are subjected to controlled by multiple genes.Capsicum genetic breeding person is according to the hereditary property of the resistance resource grasped, selects the work that conducts a research of suitable breeding technique.
At present, French INRA vegetable breeding Lefebvre of institute and Palloix (1996) are with from F in planting
1The double haploid kind of hybrid Perennial (the horizontal disease resistance of tool, India capsicum) X Yolo Wonder (susceptible, U.S.'s pimento) is a material, studies show that, 13 QTL that are distributed in several linkage groups or are in non-chain state influence the capsicum blight-resistant performance; The effect difference of these QTL has the branch of leading effect, medium effect, little effect QTL (quantitative trait locus).(1997) such as Korea Kang are material with near isogene, use the AFLP technology, and finding has 5 DNA (thymus nucleic acid) fragment only to appear in the collection of illustrative plates of anti-eqpidemic disease material.Hence one can see that, and Lefebvre and Palloix have only carried out research to the material of tool horizontal resistance, and result of study still can not be applied in the marker assisted selection; Kang etc. have only tentatively obtained the candidate AFLP mark of capsicum blight-resistant gene.
The research of external this respect still is in initial period, and present existing these still not deep enough, systems of research.
Summary of the invention
In order to overcome the deficiency that above-mentioned prior art exists, the purpose of this invention is to provide a kind of molecular marker-assisted selection method of capsicum blight-resistant breeding, thereby realize selecting quickly and efficiently in the capsicum blight-resistant breeding purpose of anti-eqpidemic disease material.Above-mentioned molecular marker-assisted selection method also can be used in capsicum blight-resistant breeding.
The present invention is at traditional capsicum blight-resistant breeding segregating generation (F
2To F
5Generation or BC
1To BC
5In the selection from generation to generation), utilize and the closely linked SCAR of capsicum blight-resistant gene (SequencedCharacterized Amplified Regions, the amplification region of sequence signatureization) two of mark primers carry out PCR (polymerase chain reaction) to the DNA of plant and detect, and the sequence of these two primers is: primer 1:5 ' aaa gct gcg gat tta gac ct 3 '; Primer 2: 5 ' aaa gct gcg gtt gaa tcg g 3 '.The renaturation temperature is 56～58 ℃ in the PCR reaction conditions.Adopt agarose gel electrophoresis technology to analyze the PCR product.From antilemic material, all can amplify the DNA band of 520 base pair sizes, from then can the not increase DNA band of these 520 base pair sizes of susceptible material.
Purpose of the present invention is achieved through the following technical solutions: a kind of molecular marker-assisted selection method of capsicum blight-resistant breeding comprises the steps:
(1) plant of capsicum blight-resistant breeding segregating generation material is seeded in 25～30 ℃, the greenhouse of relative humidity 50～70%, normal management, when treating that seedling grows 5～6 true leaves, each plant is got 1～2 spire respectively, extracts genomic dna according to the SDS method.
(2) be template with above-mentioned DNA, utilize two primers of SCAR mark to carry out pcr amplification.Two primers of described SCAR mark are respectively primer 1 and primer 2, described primer 1:5 ' aaa gct gcggat tta gac ct 3 '; Primer 2: 5 ' aaa gct gcg gtt gaa tcg g 3 '.
(3) under 56～58 ℃ annealing temperature, carry out pcr amplification, amplified production is separated ethidium bromide (adding ethidium bromide) dyeing, observation PCR result under ultraviolet lamp to final concentration 500ng/mL on 1.5% agarose gel electrophoresis.
(4) if can amplify the DNA band of 520 base pair sizes, the anti-eqpidemic disease of this material is described; The DNA band of these 520 base pair sizes if can not increase illustrates that this material is susceptible to the eqpidemic disease performance.
In order to realize the present invention better, PCR reaction cumulative volume is 25 microlitres in the described step (2), contains 10m mol/L Tris-HCl (pH8.3), 50m mol/L KCl, 1.5m mol/L MgCl
2, 0.01%gelitin, 20ng genomic dna, each primer 0.2～0.5umol/L, 0.5U Taq DNAPolymerase (TaqDNA polysaccharase).
The pcr amplification reaction parameter is in the described step (3): 94 ℃ of sex change 1min, and 58 ℃ of renaturation 1min, 72 ℃ are extended 2min, 35 circulations; Last circulation prolongs 10min for 72 ℃.
Described segregating generation is F
2To F
5Generation or BC
1To BC
5From generation to generation.
The present invention is being female parent (P from Indonesian anti-eqpidemic disease capsicum material PBC 534
1), since be male parent (P from China's northern area to the susceptible Bulgarian sharp green pepper of eqpidemic disease performance
2), with P
1, P
2And P
1* P
2, F
2Be seeded in the greenhouse for material, in the inoculation of 5～6 leaf phases, inoculum density is 10000 zoospores/ml, every strain 1.5ml.Adopt BSA (bulked segregant analysis mixes the fractional analysis method) analysis principle, adopt RAPD technology (DNA random amplified polymorphism labeling technique), obtained and the closely linked mark OPC11 of capsicum blight-resistant gene
500, with this marker clone, sequence results shows that this is labeled as 522bp.According to this sequence, two primer sequences that designed SCAR (Sequenced Characterized AmplifiedRegions, the amplification region of sequence signatureization) mark are respectively: primer 1:5 ' aaagct gcg gat tta gac ct 3 '; Primer 2: 5 ' aaa gct gcg gtt gaa tcg g 3 ', thus this RAPD mark is converted into more stable SCAR mark.
With existing capsicum blight-resistant breeding technology be artificial seedling stage the disease resistance authenticate technology compare, the present invention has following advantage and beneficial effect:
(1) the present invention can directly detect the capsicum blight-resistant gene in testing laboratory, is not subjected to the influence of envrionment conditions.
(2) utilize the present invention can in office when the phase be carried out the capsicum blight-resistant Performance Detection to the single plant in the capsicum blight-resistant breeding segregating generation, and do not influence its normal growth, and traditional method is difficult to reach.
(3) utilize the present invention to carry out the capsicum blight-resistant Performance Detection, detect rapidly, can in 1～2 day, obtain detected result, and traditional method needs 40～50 days.
(4) utilize the present invention to carry out the capsicum blight-resistant Performance Detection, expense is low, only is 10～20 yuan of/part materials, and traditional method needs 100～150 yuan of/part materials.
Description of drawings
Fig. 1 is for utilizing the SCAR mark to 12 BC
1Detection figure for strain system.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
The Chifeng's Cayenne pepper that derives from the Inner Mongol with high-quality, high yield, sense eqpidemic disease is a female parent, is male parent with the anti-eqpidemic disease material PBC830 from vegetables centre of research and development, Asia, obtains cross combination F
1, be that recurrent parent is backcrossed with the material Chifeng Cayenne pepper of high-quality, high yield, sense eqpidemic disease, BC from generation to generation obtains to backcross
1To capsicum blight-resistant breeding segregating generation BC
1When selecting:
(1) this material is seeded in 25～30 ℃, the greenhouse of relative humidity 50～70%, when treating that seedling grows 5～6 true leaves, each plant is got 1～2 spire respectively, according to (Agricultural University Of Jiangxi's journal such as Wang Deyuan (1998), 1998,20 (2): 180-183) improved SDS method is extracted genomic dna.
(2) be template with above-mentioned genomic dna, utilize the SCAR mark two primers (primer 1:5 ' aaa gct gcg gat tta gac ct 3 '; Primer 2: 5 ' aaa gct gcg gtt gaa tcg g 3 ') carry out pcr amplification.
PCR reaction cumulative volume is 25 microlitres, contains 10mmol/L Tris-HCl (pH8.3), 50mmol/LKC1,1.5mmol/L MgCl
2, 0.01%gelitin, 20ng genomic dna, each primer 0.2～0.5umol/L, 0.5U Taq DNA Polymerase (Taq archaeal dna polymerase).
(3) under 58 ℃ annealing temperature, carry out pcr amplification, reaction parameter is: 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 2min, 35 circulations; Last circulation prolongs 10min for 72 ℃.Pcr amplification reaction carries out on the 9700 type PCR instrument that PE company produces.
(4) above-mentioned amplified production is separated ethidium bromide (adding ethidium bromide) dyeing to final concentration 500ng/mL on 1.5% agarose gel electrophoresis.Observation PCR result under ultraviolet lamp.
(5) utilize this SCAR mark to 12BC
1Carried out the PCR detection for plant, found that, all can amplify the DNA band of 520 base pair sizes in the antilemic material, and susceptible material is according to (guangdong agricultural sciences such as Wang Deyuan, 2001, (2): method 37-39) is carried out artificial inoculation on seedling phytophthora blight of pepper resistance and is identified back plant death, the DNA band of these 520 base pair sizes that can not increase; During PCR detects, as Fig. 1 for utilizing the SCAR mark to 12 BC
1Detection figure for strain system.M:100bp DNA ladder (each band molecular weight be respectively 900,700,600,500,400,300bp) from top to bottom wherein; Gush 1～12 and represent 12 individual plants, the left side is gushed 1～12 and is 58 ℃ of renaturation temperature, and the right is gushed 1～11 and is 56 ℃ of renaturation temperature.
(6) with disease-resistant single-strain planting to the field, normal management is by the individual plant seed of gathering.
(7) with planting seed in the greenhouse, operate according to above-mentioned (1) to (4) step, selection can amplify the plant and the plant that can not amplify the DNA band of these 520 base pair sizes of the DNA band of 520 base pair sizes, plant, normal management is by the individual plant seed of gathering.
(8) can amplify the seed of individual plant of DNA band of 520 base pair sizes and the planting seed of individual plant of DNA band that can not amplify these 520 base pair sizes in the greenhouse, according to (guangdong agricultural sciences such as Wang Deyuan, 2001, (2): method 37-39) is carried out artificial inoculation on seedling phytophthora blight of pepper resistance and is identified.Found that the sickness rate that the strain that can amplify the DNA band of 520 base pair sizes is is 5%, shows as disease-resistant; And the sickness rate that the strain that can not amplify the DNA band of these 520 base pair sizes is is 90%, shows as susceptible.
The field is planted by disease-resistant capsicum strain system, to carry out the selection of other important characters such as quality, high yield.Human and material resources and soil have been reduced like this.
The Bulgarian sharp green pepper that derives from the northern area of China with high-quality, high yield, sense eqpidemic disease is a female parent, to be male parent from Indonesian anti-eqpidemic disease capsicum material PBC 534, obtains cross combination F
1, F
1Selfing obtains F from generation to generation
2To capsicum blight-resistant breeding segregating generation F
2When selecting:
(1) this material is seeded in 25～30 ℃, the greenhouse of relative humidity 50～70%, when treating that seedling grows 5～6 true leaves, each plant is got 1～2 spire respectively, according to (Agricultural University Of Jiangxi's journal such as Wang Deyuan (1998), 1998,20 (2): 180-183) improved SDS method is extracted genomic dna.
(2) be template with above-mentioned genomic dna, utilize the SCAR mark two primers (primer 1:5 ' aaa gct gcg gat tta gac ct 3 '; Primer 2: 5 ' aaa gct gcg gtt gaa tcg g 3 ') carry out pcr amplification.
PCR reaction cumulative volume is 25 microlitres, contains 10mmol/L Tris-HCl (pH8.3), 50mmol/LKCl, 1.5mmol/L MgCl
2, 0.01%gelitin, 20ng genomic dna, each primer 0.2～0.5umol/L, 0.5U Taq DNA Polymerase (Taq archaeal dna polymerase).
(3) under 58 ℃ annealing temperature, carry out pcr amplification, reaction parameter is: 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 2min, 35 circulations; Last circulation prolongs 10min for 72 ℃.Pcr amplification reaction carries out on the 9700 type PCR instrument that PE company produces.
(4) above-mentioned amplified production is separated ethidium bromide (adding ethidium bromide) dyeing to final concentration 500ng/mL on 1.5% agarose gel electrophoresis.Observation PCR result under ultraviolet lamp.
(5) utilize this SCAR mark to F
2Carried out the PCR detection for plant, found that, all can amplify the DNA band of 520 base pair sizes in the antilemic material, and susceptible material is according to (guangdong agricultural sciences such as Wang Deyuan, 2001, (2): method 37-39) is carried out artificial inoculation on seedling phytophthora blight of pepper resistance and is identified back plant death, the DNA band of these 520 base pair sizes that can not increase.
(6) with disease-resistant single-strain planting to the field, normal management by the individual plant seed of gathering, obtains F
3From generation to generation.
(7) with planting seed in the greenhouse, operate according to above-mentioned (1) to (4) step, selection can amplify the plant and the plant that can not amplify the DNA band of these 520 base pair sizes of the DNA band of 520 base pair sizes, plant, normal management is by the individual plant seed of gathering.
(8) can amplify the seed of individual plant of DNA band of 520 base pair sizes and the planting seed of individual plant of DNA band that can not amplify these 520 base pair sizes in the greenhouse, according to (guangdong agricultural sciences such as Wang Deyuan, 2001, (2): method 37-39) is carried out artificial inoculation on seedling phytophthora blight of pepper resistance and is identified.Found that the sickness rate that the strain that can amplify the DNA band of 520 base pair sizes is is 8%, shows as disease-resistant; And the sickness rate that the strain that can not amplify the DNA band of these 520 base pair sizes is is 85%, shows as susceptible.
The field is planted by disease-resistant capsicum strain system, to carry out the selection of other important characters such as quality, high yield.Human and material resources and soil have been reduced like this.
Utilize the present invention to carry out the capsicum blight-resistant marker assisted selection, from the DNA extraction to the detected result, come out to need 1～2 day, spend lower.
SEQUENCELISTING
＜110〉Vegetables Inst., Guangdong Academy of Agricultural Sciences
＜120〉a kind of molecular marker-assisted selection method of capsicum blight-resistant breeding
<130>26
<160>2
<170>PatentIn?version?3.2
<210>1
<211>20
<212>DNA
<213>Artificial?sequence
<220>
＜223〉utilize the primer 1 of SCAR mark
<400>1
aaagctgcgg?atttagacct 20
<210>2
<211>19
<212>DNA
<213>Artificial?sequence
<220>
＜223〉utilize the primer 2 of SCAR mark
<400>2
aaagctgcgg?ttgaatcgg 19
Claims (4)
1, a kind of molecular marker-assisted selection method of capsicum blight-resistant breeding is characterized in that comprising the steps:
(1) plant of capsicum blight-resistant breeding segregating generation material is seeded in 25～30 ℃, the greenhouse of relative humidity 50～70%, when treating that seedling grows 5～6 true leaves, each plant is got 1～2 spire respectively, extracts genomic dna according to the SDS method;
(2) be template with above-mentioned DNA, utilize two primers of SCAR mark to carry out pcr amplification, two primers of described SCAR mark are respectively primer 1 and primer 2,
Primer 1:5 ' aaa gct gcg gat tta gac ct 3 ';
Primer 2: 5 ' aaa gct gcg gtt gaa tcg g 3 ';
(3) under 56～58 ℃ annealing temperature, carry out pcr amplification, amplified production is separated ethidium bromide staining, observation PCR result under ultraviolet lamp on 1.5% agarose gel electrophoresis;
(4) if amplify the DNA band of 520 base pair sizes, the anti-eqpidemic disease of this material is described; The DNA band of these 520 base pair sizes if can not increase illustrates that this material is susceptible to the eqpidemic disease performance.
2, the molecular marker-assisted selection method of a kind of capsicum blight-resistant breeding according to claim 1, it is characterized in that: PCR reaction cumulative volume is 25 microlitres in the described step (2), contain 10m mol/LTris-HCl, 50m mol/L KCl, 1.5m mol/L MgCl
2, 0.01%gelitin, 20ng genomic dna, each primer 0.2～0.5u mol/L, 0.5U Taq archaeal dna polymerase.
3, the molecular marker-assisted selection method of a kind of capsicum blight-resistant breeding according to claim 1 is characterized in that: the pcr amplification reaction parameter is in the described step (3): 94 ℃ of sex change 1min, and 58 ℃ of renaturation 1min, 72 ℃ are extended 2min, 35 circulations; Last circulation prolongs 10min for 72 ℃.
4, the molecular marker-assisted selection method of a kind of capsicum blight-resistant breeding according to claim 1 is characterized in that: described segregating generation is F
2To F
5Generation or BC
1To BC
5From generation to generation.
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| CN101487048B (en) * | 2009-01-19 | 2012-05-23 | 西北农林科技大学 | Molecular marker method for identifying pepper anti-epidemic disease character |
| CN102812836B (en) * | 2012-08-20 | 2013-09-11 | 西北农林科技大学 | Method for rapidly identifying functions of related disease resisting genes of hot pepper |
| CN107190094B (en) * | 2017-07-24 | 2020-10-27 | 中国农业大学 | Application of pepper molecular marker and polymorphism thereof in identifying pepper pollen fertility |
| CN113785741A (en) * | 2021-11-18 | 2021-12-14 | 山东寿光蔬菜种业集团有限公司 | Seed production method for pepper breeding |
| CN115927699B (en) * | 2022-07-18 | 2024-01-26 | 江西省农业科学院蔬菜花卉研究所 | Molecular marker for identifying germplasm of pepper with epidemic disease resistance and application thereof |
| CN116439135A (en) * | 2023-05-09 | 2023-07-18 | 遵义职业技术学院 | Quick cultivation method for capsicum by using molecular markers |
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Non-Patent Citations (6)
| Title |
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| 几个抗疫病性不同的辣椒材料抗病基因同源序列的分离与比较. 易永图等.园艺学报，第30卷第5期. 2003 |
| 几个抗疫病性不同的辣椒材料抗病基因同源序列的分离与比较. 易永图等. 园艺学报，第30卷第5期. 2003 * |
| 利用差异显示技术克隆辣椒抗疫病相关DNA序列的研究. 易永图等.中国蔬菜，第12期. 2005 |
| 利用差异显示技术克隆辣椒抗疫病相关DNA序列的研究. 易永图等. 中国蔬菜，第12期. 2005 * |
| 辣椒SRAP-PCR反应体系的建立与优化. 任，羽等.分子植物育种，第2卷第5期. 2004 |
| 辣椒SRAP-PCR反应体系的建立与优化. 任羽等. 分子植物育种，第2卷第5期. 2004 * |
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