CN102747070A - Two CAPs markers tightly linked with muskmelon anti-powdery mildew gene Pm-AN and its application method - Google Patents
Two CAPs markers tightly linked with muskmelon anti-powdery mildew gene Pm-AN and its application method Download PDFInfo
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- CN102747070A CN102747070A CN2011101015049A CN201110101504A CN102747070A CN 102747070 A CN102747070 A CN 102747070A CN 2011101015049 A CN2011101015049 A CN 2011101015049A CN 201110101504 A CN201110101504 A CN 201110101504A CN 102747070 A CN102747070 A CN 102747070A
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Abstract
The invention relates to two codominance CAPs molecular markers which are positioned at two sides of muskmelon anti-powdery mildew gene Pm-AN and tightly linked with Pm-AN, and its application method, which belongs to the biology technical field. The markers are used for muskmelon anti-powdery mildew molecular markers for auxiliary breeding, and breeding purpose and breeding efficiency of the muskmelon anti-powdery mildew breeding can be substantially enhanced. The application of the markers is not influenced by disease factors in fields, can effectively select pure disease resistance gene type and heterozygosis disease resistance gene type individuals, and the recombinant genotype individuals are generated at two sides of the anti-powdery mildew gene. The markers can be used for disease resistance molecule polymerization breeding without transgene security problem.
Description
Technical field
The present invention relates to two and be positioned at muskmelon mildew-resistance gene Pm-AN both sides, and with closely linked two codominance CAPs molecule markers of Pm-AN and method of use, belong to biological technical field.
Background technology
The generalized molecule marker is meant heritable and detectable dna sequence dna or protein.The molecule marker of narrow sense only is meant dna marker, is the direct reflection of genetic polymorphism on the dna level, and general alleged molecule marker notion promptly is defined within this category.But the biological heritable variation that in the long-term evolution process, produces all is because due to the variation of DNA base sequence.There is very rich DNA base sequence variation in the same species, for the development and utilization molecule marker has been established solid foundation.
Dna molecular marker is meant that biological gene group DNA combines to wait measure processing rear electrophoresis, the DNA fragment specific of detected certain variation features of reflection genome through digestion with restriction enzyme, polymerase chain reaction (PCR) amplification or both.
Molecule marker is compared with other genetic markers, has following advantage: (1) directly with the form performance of DNA, does not receive the influence of developmental stage, histoorgan, envrionment conditions; (2) numbers of poles is many, almost spreads all over whole genome; (3) loci of molecule marker variation level is much abundanter than phenotypic markers, and promptly polymorphum is very high, so that need not create special genetic stocks specially; (4) molecule marker does not have detrimentally affect usually to organism, does not influence the performance of biological character yet, promptly shows as " neutrality "; (5) many molecule markers are the codominant marker, can identify homozygous genotype and heterozygous genes type, and complete genetic information is provided; (6) contain much information, analysis efficiency is high; (7) genetic stability, good reliability, favorable reproducibility; (8) speed is fast, and is easy and simple to handle.These advantages of molecule marker make it in the theory of life science and applied research, have extremely important using value.In recent years; Molecular biology undergoes an unusual development rapidly; Attract people's attention especially with the closely-related molecule marker of Crop Genetic Breeding, make that " the selecting indirectly " in the plant breeding become possibility, improved the accuracy of genetic analysis and the validity of seed selection kind widely; More and more coming into one's own in the genetic breeding field, is the trend of breeding work from now on the two combination.Molecular marking technique commonly used at present mainly contains RFLP, RAPD, AFLP, SSR, SNP, SCAR, SRAP, STS, EST etc.
CAPs (cleaved amplification polymorphism sequence-tagged sites) CAPs technology is called PCR-RFLP again.When being the PCR primer amplification target material with special design, because the base mutation of specific site, insertion or disappearance number are seldom, so that no polymorphic appearance often need be carried out enzyme to corresponding pcr amplified fragment and cut processing, to detect its polymorphum.CAPs is marked in the diplont research can bring into play enormous function.Thereby the advantage of this mark at first is a codominance can distinguish homozygote and heterozygote genotype, secondly is very high stability and safety.
Melon powdery mildew is one of main disease of muskmelon, and cultivating the mildew-resistance melon variety is the important goal of muskmelon breeding for disease resistance.Xinjiang is the secondary origin center of thick-skinned melon, is the most susceptible Powdery Mildew of China's thick-skinned melon variety of representative with the hami melon.The method of traditional breeding method is difficult to distinguish isozygotys and the heterozygous genes type, is difficult to realize the polymerization of different mildew-resistance genes, more can't be chosen near the individuality that reorganization takes place of disease-resistant gene.The invention provides and introduce each CAPs molecule marker of No. two mildew-resistance gene Pm-AN both sides of disease-resistant variety peace farming; Carry out assistant breeding through these two with the closely linked mark of mildew-resistance gene Pm-AN and overcome above-mentioned difficulties, can improve the breeding efficiency of muskmelon mildew-resistance new variety greatly.
Summary of the invention
The objective of the invention is to invent a kind of muskmelon mildew-resistance gene chain codominance molecule marker and method of use; The individual disease-resistant or susceptible two kinds of genotype of muskmelon be can judge through the molecule marker among the present invention, muskmelon mildew-resistance individual selection purpose and efficiency of selection therefore improved.Be used for muskmelon mildew-resistance molecular mark.
For realizing this purpose, the present invention takes following technical scheme:
With closely linked two the CAPs molecule markers of mildew-resistance gene Pm-AN, it is characterized in that having SEQ ID NO:1 in the sequence table～8 described dna sequence dnas.
SEQ ID NO:1 is the sequence of the upstream primer RPW1 of the closely linked CAPs mark of mildew-resistance gene Pm-AN RPW in the sequence table.
SEQ ID NO:2 is the sequence of the downstream primer RPW2 of the closely linked CAPs mark of mildew-resistance gene Pm-AN RPW in the sequence table.
SEQ ID NO:3 is the segmental dna sequence dna of the specific amplified of the closely linked CAPs mark of mildew-resistance gene Pm-AN RPW in disease-resistant variety in the sequence table.
SEQ ID NO:4 is the segmental dna sequence dna of the specific amplified of the closely linked CAPs mark of mildew-resistance gene Pm-AN RPW in susceptible variety in the sequence table.
SEQ ID NO:5 is the sequence of the upstream primer MRGH63B1 of the closely linked CAPs mark of mildew-resistance gene Pm-AN MRGH63B in the sequence table.
SEQ ID NO:6 is the sequence of the upstream primer MRGH63B2 of the closely linked CAPs mark of mildew-resistance gene Pm-AN MRGH63B in the sequence table.
SEQ ID NO:7 is the segmental dna sequence dna of the specific amplified of the closely linked CAPs mark of mildew-resistance gene Pm-AN MRGH63B in disease-resistant variety in the sequence table.
SEQ ID NO:8 is the segmental dna sequence dna of the specific amplified of the closely linked CAPs mark of mildew-resistance gene Pm-AN MRGH63B in susceptible variety in the sequence table.
With closely linked two the CAPs molecule markers of mildew-resistance gene Pm-AN, its method of use may further comprise the steps:
Get the disease-resistant and susceptible hybridization of muskmelon and separate the individual organization material of offspring; Extract the muskmelon genomic dna; Use RPW and MRGH63B specificity amplification primer that the muskmelon genomic dna is carried out pcr amplification; Two CAPs mark amplified fragments obtain gene order respectively shown in sequence table SEQ ID NO:3～4 and SEQ ID NO:7～8 through order-checking in disease-resistant variety and the susceptible variety; Use restriction enzyme A lu I and Msp I that amplified production is carried out enzyme and cut, enzyme is cut product carry out agarose gel electrophoresis, obtain the DNA band different with the height shown in the accompanying drawing 2 like accompanying drawing 1.Said gene group DNA extraction method, PCR reaction system and condition, endonuclease reaction system and condition are said with embodiment.Therefore disease-resistant specific fragment two less bands occur owing to cut by restriction endonuclease.Susceptible specific fragment can not be cut by restriction endonuclease, therefore occurs one than big band.Can judge the susceptible or disease-resistant gene type genotype that muskmelon is individual according to this result, also can judge between disease-resistant gene and CAPs mark RPW and MRGH63B and whether recombinate.Thereby carry out the molecular marker assisted selection breeding.
The invention has the advantages that: 1. directly with the form performance of DNA, do not receive the influence of field Powdery Mildew morbidity.2. molecule marker is the codominant marker, can identify homozygous genotype and heterozygous genes type, and complete genetic information is provided.3. use this marker assisted selection muskmelon mildew-resistance kind and can improve efficiency of selection greatly.4. can use this mark to realize different mildew-resistance gene molecule aggregations.5. the individuality through selecting to take place between mildew-resistance gene Pm-AN and two the closely linked molecule markers to recombinate can be broken the chain of mildew-resistance gene and unfavorable gene, avoids chain burden.6. detect test-results through agarose gel electrophoresis, the polyacrylamide gel electrophoresis that need not complicated operation detects.
Description of drawings
Fig. 1 is that mark PRW is at two parents and F
2The agarose gel electrophoresis figure that Different Individual detects, the arrow indication is the electrophoretic band size among the figure.
Fig. 2 is that mark MRGH63B is at two parents and F
2The agarose gel electrophoresis figure that Different Individual detects, the arrow indication is the electrophoretic band size among the figure.
Embodiment
Embodiment is through the chain molecule marker of the muskmelon mildew-resistance gene Pm-AN disease-resistant strain system of selecting to isozygoty
1. experiment material
No. two, muskmelon mapping parent material peace farming and hami melon K413 are provided by the melon Engineering Technical Research Centre of Xinjiang country.No. two, peace farming is taught in 1986 by Lin Depei and is introduced from Japan, good quality and high output, and multiple diseases such as mildew-resistance, dead arm, virus disease, oidium and blight, fruit is rounded, and white skin plain boiled pork has reticulate pattern, the small fruit type thick-skinned melon (15cm * 12cm).K413 is that typical hami melon type muskmelon plant disease resistance is poor, fruit ellipse, and the yellow meat of Calusena lansium, mouthfeel is crisp, the big (30cm * 16cm) of melon type.
2. experimental technique
2.1 the extraction of Xinjiang muskmelon genomic dna
(1) get the fresh muskmelon blade in the 50mg left and right sides in EP pipe, add 2 * CTAB damping fluid of 0.4ml, with grinding, 65 ℃ of water-bath 0.5h, during shake up gently 1 time;
(2) take out centrifuge tube and be chilled to room temperature, add isopyknic chloroform/primary isoamyl alcohol (24: 1), the light and slow mixing of putting upside down;
(3) the centrifugal 10min of 12000r/min under the room temperature;
(4) supernatant is changed in another centrifuge tube, add the Virahol of equal-volume precooling, put upside down mixing;
(5) the centrifugal 10min of 12000r/min abandons supernatant;
(6) deposition is dissolved in the 20 μ l distilled waters-20 ℃ of preservations.
2.2DNA the pcr amplification of template
The RPW mark and MRGH63B labeled primer sequence:
RPW1 (primer 1): GCATTGGGTGTTCCGTTTA
RPW2 (primer 2): CAGTTGGGTAATCGGGAG
MRGH63B1 (primer 1): GAACATCTCATCCCTCAAGTT
MRGH63B2 (primer 2): GGAAGAACAGAGCCAAAGAA
Reaction TV 20 μ l, system is following.
Amplification PCR response procedures is following in advance:
After the PCR reaction finishes, get the preparatory amplified production of 6 μ l, 1% agarose gel electrophoresis detects preparatory expanding effect.Amplified production is through ddH in advance
2O dilution back is as the template of following selective amplification.
2.3 the enzyme of sample DNA is cut
Respectively with kind of a restriction enzyme A luI, Msp I to the RPW mark and the PCR reaction product of MRGH63B mark carry out enzyme and cut, reaction volume is 10 μ l, 37 ℃ of water-bath 4h behind the mixing.
After reaction finished, whether 1.5% agarose gel electrophoresis detected enzyme and cuts complete.
3. outcome record and analysis
The genomic dna that contains No. two, the peace farming of mildew-resistance gene Pm-AN carries out pcr amplification with RPW1 and RPW2 primer; Product is cut the band that produces 265bp and 380bp through the AluI enzyme, and susceptible variety K413 genome PCR product produces the band of 645bp after Alu I enzyme is cut.
The genomic dna that contains No. two, the peace farming of mildew-resistance gene Pm-AN carries out pcr amplification with MRGH63B1 and MRGH63B2 primer; Product is cut the band that produces 139bp and 103bp through Msp I enzyme, and K413 genome PCR product produces the band of 242bp after Msp I enzyme is cut.
At No. two, peace farming and 143 F of K413 hybridization
2In the individuality; Find that 2 strains recombinate between RPW mark and powdery mildew gene site, a strain RPW mark only has the 645bp band, i.e. performance susceptible parent's the genotype of isozygotying; Plant is disease-resistant; Therefore this plant has mildew-resistance gene, and in the very near distance of mildew-resistance gene one side, reorganization has taken place, and can be used to break the chain burden of unfavorable gene; Another strain performance RPW mark shows as the heterozygote genotype, and plant shows susceptible.All the other 141 strain RPW marker detection results are consistent with the disease resistance performance.
At No. two, peace farming and 143 F of K413 hybridization
2In the individuality; Find that 2 strains recombinate between RPW mark and powdery mildew gene site, a strain RPW mark only has the 240bp band, i.e. performance susceptible parent's the genotype of isozygotying; Plant is disease-resistant; Therefore this plant has mildew-resistance gene, and in the very near distance of mildew-resistance gene one side, reorganization has taken place, and can be used to break the chain burden of unfavorable gene; Another strain performance RPW mark shows as the heterozygote genotype, and plant shows susceptible.All the other 141 strain RPW marker detection results are consistent with the disease resistance performance.
This zone shows that through the genetic linkage mapping analysis RPW mark and MRGH63B mark lay respectively at mildew-resistance gene Pm-AN both sides 1.4cM and 2cM.Using two marks that separation offspring genotype is carried out judging nicety rate simultaneously is 100%.No. two, peace farming and 143 F of K413 hybridization
2In the individuality, use two codominant markers to identify disease-resistant individuality totally 36 strains of isozygotying, disease-resistant separation does not take place in the self progeny, and these individualities are used for seed selection mildew-resistance new germ plasm and new variety.
Claims (5)
1. the codominance CAPs molecule marker RPW of muskmelon mildew-resistance is characterized in that: use in the sequence table acquisition of can increasing of the described dna primer sequence in SEQ ID NO:1～2, comprise the gene order of SEQ ID NO:3～4.
2. the codominance CAPs molecule marker MRGH63B of muskmelon mildew-resistance is characterized in that: use in the sequence table acquisition of can increasing of the described dna primer sequence in SEQ ID NO:5～6, comprise the gene order of SEQ ID NO:7～8.
3. molecule marker as claimed in claim 1 is characterized in that: carry out the product of pcr amplification through primer SEQ ID NO:1～2, cut at the mildew-resistance material polymorphic with sense white powder material production through Alu I enzyme.
4. molecule marker as claimed in claim 2 is characterized in that: carry out the product of pcr amplification through primer SEQ ID NO:5～6, cut at the mildew-resistance material polymorphic with sense white powder material production through the MspI enzyme.
5. like the method for use of claim 1 and the described molecule marker of claim 2; It is characterized in that: through extracting the muskmelon tissue DNA, carry out pcr amplification, cut with restriction enzyme A lu I and Msp I enzyme with the primer sequence of SEQID NO:1～2 and SEQ ID NO:5～6; Agarose gel electrophoresis detects; Judge whether plant contains mildew-resistance gene Pm-AN, and whether genotype isozygotys, and whether recombinate between two muskmelon CAPs molecule markers of these gene both sides.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630215A (en) * | 2015-01-23 | 2015-05-20 | 北京市农林科学院 | Series molecular markers closely linked with muskmelon powdery mildew resistant gene Pm-2Fand acquisition method of series molecular markers |
| CN107164481A (en) * | 2017-06-01 | 2017-09-15 | 中国农业科学院蔬菜花卉研究所 | Powdery mildew of melon correlation SSR marker and its application |
| CN109355419A (en) * | 2018-11-20 | 2019-02-19 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the downstream tobacco TMV resistant gene N (3 ' end) Linkage drag and its application |
| CN109439789A (en) * | 2018-11-20 | 2019-03-08 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the upstream tobacco TMV resistant gene N (5 ' end) Linkage drag and its application |
| CN110055351A (en) * | 2019-05-31 | 2019-07-26 | 东北农业大学 | One kind molecular labeling BanII-1 relevant to melon powdery mildew resistance and its application |
| CN111424109A (en) * | 2020-05-14 | 2020-07-17 | 上海市农业科学院 | SNP molecular marker for identifying powdery mildew resistance traits of melons and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630215A (en) * | 2015-01-23 | 2015-05-20 | 北京市农林科学院 | Series molecular markers closely linked with muskmelon powdery mildew resistant gene Pm-2Fand acquisition method of series molecular markers |
| CN107164481A (en) * | 2017-06-01 | 2017-09-15 | 中国农业科学院蔬菜花卉研究所 | Powdery mildew of melon correlation SSR marker and its application |
| CN107164481B (en) * | 2017-06-01 | 2022-12-13 | 中国农业科学院蔬菜花卉研究所 | Powdery mildew resistance related SSR (simple sequence repeat) marker for muskmelon and application of powdery mildew resistance related SSR marker |
| CN109355419A (en) * | 2018-11-20 | 2019-02-19 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the downstream tobacco TMV resistant gene N (3 ' end) Linkage drag and its application |
| CN109439789A (en) * | 2018-11-20 | 2019-03-08 | 云南省烟草农业科学研究院 | One group of molecular labeling for breaking the upstream tobacco TMV resistant gene N (5 ' end) Linkage drag and its application |
| CN109355419B (en) * | 2018-11-20 | 2021-05-18 | 云南省烟草农业科学研究院 | Group of molecular markers for breaking N downstream (3' end) linkage drag of tobacco TMV resistance gene and application thereof |
| CN110055351A (en) * | 2019-05-31 | 2019-07-26 | 东北农业大学 | One kind molecular labeling BanII-1 relevant to melon powdery mildew resistance and its application |
| CN111424109A (en) * | 2020-05-14 | 2020-07-17 | 上海市农业科学院 | SNP molecular marker for identifying powdery mildew resistance traits of melons and application thereof |
| CN111424109B (en) * | 2020-05-14 | 2023-04-14 | 上海市农业科学院 | SNP molecular marker for identifying powdery mildew resistance traits of melons and application thereof |
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