CN104087579A - Molecular marker closely linked with corn bacterial wilt resistance genes and primer and application thereof - Google Patents
Molecular marker closely linked with corn bacterial wilt resistance genes and primer and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker which is closely linked with corn bacterial wilt resistance genes. The molecular marker is a nucleotide sequence as shown in SEQ ID NO.1 in a sequence table or a nucleotide sequence as shown in SEQ ID NO.2 in the sequence table. A primer pair for amplifying the molecular marker is a nucleotide sequence as shown in SEQ ID NO.3 in the sequence table and a nucleotide sequence as shown in SEQ ID NO.4 in the sequence table. When the molecular marker is applied to corn bacterial wilt detection, amplification is performed through PCR, if an obtained PCR amplification product is a 234bp basic group fragment, the corn to be detected is of a bacterial wilt resistant variety, and if the obtained PCR amplification product is a 274bp alkali group fragment, the corn to be detected is of a bacterial wilt infected variety. By adopting the method, whether the corn has the bacterial wilt resistance or not can be detected before the corn is seeded, selective seeding can be achieved, the breeding time is shortened, and the method is rapid, simple and convenient and can be widely applied to assisted breeding of corn.
Description
Technical field
The invention belongs to agricultural biological technical field, be specifically related to method and PCR primer and application thereof for detection of Corn Stalk Rot resistance.
Background technology
There is the shortcomings such as the low and breeding cycle of efficiency of selection is long in the conventional breeding method based on Phenotypic Selection, in the urgent need to injecting molecular approach means, auxiliary in genotype orthoselection expeditiously, could cultivate quickly and efficiently excellent corn variety.Along with developing rapidly of molecular biology and genomics, the application of molecular marking technique is more extensive.The molecule marker of PCR-based as micro-satellite or SSR (simple sequence repeat) etc. have that polymorphic rate is high, relatively stable, detection method is easy fast and the feature such as easy handling and being widely used.Because molecular marker assisted selection is not subject to such environmental effects and the aobvious recessive interference of proterties etc., can be from molecular level orthoselection objective trait gene, also likely break chain between unfavorable gene simultaneously and expeditiously the multiple excellent genes of polymerization in one.Usually said molecule marker had both comprised that the linked marker of target gene also comprised target gene self functional label.The auxiliary multiple gene polymerization breeding technique of molecule marker has become a kind of development trend of the crop breeding such as corn, paddy rice research, use this technology can by high-quality, how the gene pyramiding such as anti-and high yield is in minority key variety, can key be to obtain the practical molecule marker with objective trait gene or main effect QTL compact linkage.
Corn Stalk Rot claims again corn stalk rot disease, Causal Organism of Maize Basal Stalk etc., have morbidity suddenly, development rapidly, the underproduction is serious, prevent and treat difficult feature, is one of the Major Diseases in corn producing region, the world.In recent years, Corn Stalk Rot generally occurs in corn seed producing field, China various places, land for growing field crops, general time production loss is in 20% left and right, serious reaches more than 50%, the degree that caused harm in recent years obviously increases the weight of, and is the trend increasing the weight of year by year, so research pathogen of maize stalk rot and occurrence regularity, cultivate disease-resistant variety, reduce corn yield loss significant.
(the Yang Q. such as Yang, Yin G.M., Guo Y.L., et al.A major QTL for resistance to Gibberella stalk rot in maize[J] .theor Appl Genet.2010.121:673-687.) with the F of self-mating system 1145 × Y331
2, BC
2f
1and BC
3f
1colony, as Research Group, confirms that qRfg1 is anti-stem rot main effect QTL, by following the trail of BC
4f
1to BC
6f
1the Fine Mapping research in generation, finds qRfg1 and mark SSR334 and SSR58 close linkage, and distance is 0-500bp, and resistance contribution rate is 32-43%, and thinks that qRfg1 can significantly improve the anti-stem rot ability of corn by performance hereditary potency.(the Qing Yang such as Qing, Zhi Li, et al.CACTA-like transposable element in ZmCCT attenuated photoperiod sensitivity and accelerated the postdomestication spread of maize.PNAS 2013, 10.) report that this disease-resistant QTL is a gene that comprises CCT structural domain, called after ZmCCT, this genoid that comprises CCT structural domain may further affect by affecting the photoperiod other crucial economical characters of farm crop, be one and have great value, can be applicable to the key gene of crop genetic improvement.
Summary of the invention
The object of the invention is to be provided for detecting specific Function molecule marker and the method for Corn Stalk Rot resistance, adopt the inventive method can before corn seeding, detect corn and whether there is bacterial wilt resistance, sow selectively, shorten breeding time, the inventive method is fast and convenient, can be widely used in the assistant breeding of corn.
Adopt following technical scheme for realizing the object of the invention:
One and the closely linked molecule marker of Corn Stalk Rot resistant gene, is characterized in that, described molecule marker is nucleotide sequence shown in the nucleotide sequence shown in sequence table SEQ ID NO:1 or sequence table SEQ ID NO:2.
Described in the claim 1 that increases, a primer pair for molecule marker, is characterized in that, the primer 1 of described primer pair is the nucleotide sequence shown in sequence table SEQ ID NO:3, and the primer 2 of described primer pair is the nucleotide sequence shown in SEQ ID NO:4.
A method that detects Corn Stalk Rot resistance, is characterized in that, described method comprises the steps:
(1) pcr amplification: the genome extracting using corn to be measured, as DNA cloning template, consists of amplimer to carrying out pcr amplification with nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:3 and sequence table SEQ ID NO:4;
(2) qualification of amplified production: if gained pcr amplification product is 234bp base fragment, treat that corn is resistance to bacterial wilt kind, if gained pcr amplification product is 274bp base fragment, corn to be measured is bacterial wilt susceptible variety.
4, a kind of method that detects Corn Stalk Rot resistance according to claim 3, is characterized in that, in described step (2), 234bp base fragment is the nucleotide sequence shown in sequence table SEQ ID NO:1; 274bp base fragment is nucleotide sequence shown in sequence table SEQ ID NO:2.
The genome method that corn to be measured in described step (1) extracts is alkaline-heating method, concrete steps are: get 1/20 corn kernel endosperm 0.2Mol/L sodium hydroxide 40ml 100 DEG C of water-baths 5 minutes, add again the 0.17Mol/Ltris-HCL water-bath 3 minutes of 60ml, 4 DEG C of refrigerators are cooling for subsequent use, obtain the genome that described corn to be measured extracts.
The reaction system of the pcr amplification in described step (1) is: containing 10X Buffer 2 μ 1 of Mg2+, the dNTP 0.4 μ l of 1OmM, each 2 μ 1 of nucleotide sequence shown in nucleotide sequence shown in the sequence table SEQ ID NO:3 of 5 μ M and sequence table SEQ ID NO:4, genomic dna 1 μ 1 that described corn to be measured extracts, taq enzyme 0.5 μ 1 of 5U/ μ 1, all the other are ultrapure water, and reaction volume is 20 μ 1, drip a mineral oil and cover; PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 1Omin.
The application of described molecule marker in corn breeding.
The application of described primer pair in corn breeding.
The application of the method for described detection Corn Stalk Rot resistance in corn breeding.
Described molecule marker is positioned at the promoter region of resistant gene ZmCCT.
The present invention has following beneficial effect with respect to prior art:
Method of the present invention has overcome in conventional breeding because Phenotypic Selection is subject to environmental factors to be disturbed and causes the problems such as breeding efficiency is low, can utilize the primer special of the composition of nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:3 and sequence table SEQ ID NO:4 to Corn Stalk Rot resistant gene is carried out early generation selection and shortens breeding cycle, thereby filter out quickly the corn material of resistance to bacterial wilt.The present invention can be applicable to corn breeding, to improve breeding efficiency and yield levels of maize.
Method of the present invention has overcome in molecular mark, because molecule marker and resistance to bacterial wilt gene have certain genetic distance, has the problem of the possibility separating in breeding process.The marker site of the nucleotide sequence shown in the nucleotide sequence shown in the sequence table SEQ ID NO:3 in the present invention and sequence table SEQ ID NO:4 is positioned at the promoter region of resistant gene, with resistant gene close linkage, there will not be separation, can more effectively improve breeding efficiency.
The primer pair that nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:3 of the present invention and sequence table SEQ ID NO:4 the forms genome that the corn to be measured that obtains through simple process extracts that can increase well, overcome in prior art and cannot the increase problem of the Maize genome to be measured extracting through the inventive method of this gene closely linked other molecule markers, thereby make the inventive method to carry out rapid gene evaluation and screening to the maize seed grain before sowing, then sowing selectively, reduce recruitment land used, shorten breeding cycle, further improve breeding efficiency.
Brief description of the drawings
After Fig. 1 is the primer pair amplification disease-resistant variety 1145 and not anti-kind 0908 with nucleotide sequence composition shown in nucleotide sequence shown in sequence table SEQ ID NO:5 and sequence table SEQ ID NO:6, the contrast of amplified production base fragment.
In figure, dashed region represents the disappearance of 41bp, and query is 1145 male parents, the sequence of high resistance to bacterial wilt, and query is 0908 female parent, not the sequence of resistance to bacterial wilt.
Fig. 2 is the primer pair amplification disease-resistant variety 1145 of the composition of nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:3 and sequence table SEQ ID NO:4 and the electrophorogram of not anti-kind 0908 and both cross-fertilize seed.
In figure 1145,0908 and each 3 repetitions of the each material of cross-fertilize seed, M size is 250bp.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, buys and obtains by routine biochemistry reagent company.
Embodiment 1, the acquisition of bacterial wilt resistance trait qualification primer special to the nucleotide sequence shown in the nucleotide sequence shown in sequence table SEQ ID NO:3 and sequence table SEQ ID NO:4
One, parents' genome amplification
The parents that are used for creating corn RIL (recombinant inbred line, RIL) colony be respectively 1145 and our company's seed selection 0908.0908 is maternal, not resistance to bacterial wilt; 1145 is male parent, high resistance to bacterial wilt.
Adopt CTAB method to extract the genomic dna of parents' blade, with Qing Yang (Qing Yang, Zhi Li, et al.CACTA-like transposable element in ZmCCT attenuated photoperiod sensitivity and accelerated the postdomestication spread of maize.PNAS 2013, 10. the primer pair of) announcing, formed by the nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:5 and sequence table SEQ ID NO:6, after entrusting Beijing Zi Xi biotech firm synthetic, the experiment of increasing.PCR reaction system is: 10X Buffer 2 μ l are (containing Mg2
+), dNTP 0.4 μ 1 (10mM), each 2 μ 1 of SEQ ID NO:6 shown in SEQ ID NO:5 shown in sequence table and sequence table (5 μ Μ), (40ng/ μ l) for genomic dna 1 μ l, taq enzyme 0.5 μ 1 (5 υ/μ 1), all the other are ultrapure water, and reaction volume is 20 μ 1, drip a mineral oil and cover.PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 1Omin.
Pcr amplification product is sent to order-checking, and order-checking company is Beijing Zi Xi biotech firm, and sequencing result is shown in Fig. 1 at the online comparison result of NCBI website.In table, query is 1145 male parents, the sequence of high resistance to bacterial wilt; Query is 0908 female parent, not the sequence of resistance to bacterial wilt.
Two, primer synthesizes and checking
According to the order-checking comparison result of Fig. 1, there is 41bp insertion and deletion difference zone design primer in two sequences of leap, upstream primer is nucleotide sequence shown in sequence table SEQ ID NO:3, downstream primer is nucleotide sequence shown in sequence table SEQ ID NO:4.Entrust Beijing Zi Xi biotech firm synthetic.Upstream primer and downstream primer (by this primer pair called after QK1) are positioned at gene ZmCCT promoter region.
Utilize new synthetic primer amplification 1145 and 0908, and both cross-fertilize seed.Adopt alkaline-heating method to extract corn kernel DNA, concrete steps are: get 1/20 corn kernel endosperm 0.2Mol/L sodium hydroxide 40ml 100 DEG C of water-baths 5 minutes, add again the 0.17Mol/Ltris-HCL water-bath 3 minutes of 60ml, cooling for subsequent use, obtain the genome that corn to be measured extracts.
PCR reaction system is: 10X Buffer 2 μ 1 (containing Mg2+), dNTP 0.4 μ 1 (1OmM), each 2 μ 1 of upstream primer and downstream primer (5 μ M), genomic dna 1 μ 1 that corn to be measured extracts, taq enzyme 0.5 μ 1 (5U/ μ 1), reaction volume is 20 μ 1, drips a mineral oil and covers.PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 1Omin.
Pcr amplification product 6% Polyacrylamide Gel Electrophoresis, rapid silver staining dyes and observes and take pictures, and sees Fig. 2.Pcr amplification product is checked order, can clearly find out from the electrophorogram of QK1 amplified production, 1145 have an electrophoresis band, are 234bp base fragment through amplified production corresponding to the described electrophoresis band that checks order, and are the nucleotide sequence shown in sequence table SEQ ID NO:1; 0908 has an electrophoresis band, is 274bp base fragment through amplified production corresponding to the described electrophoresis band that checks order, and is nucleotide sequence shown in sequence table SEQ ID NO:2; Hybrid has two electrophoresis bands, is 234bp and 274bp base fragment through amplified production corresponding to the described electrophoresis band that checks order, and is nucleotide sequence shown in the nucleotide sequence shown in sequence table SEQ ID NO:1 and sequence table SEQ ID NO:2.
The dependency checking of the amplified production of embodiment 2, QK1 and Corn Stalk Rot resistance trait
With high resistance to bacterial wilt material 1145 and not resistance to bacterial wilt material 0908 be parent, both hybridization after 96 F
2be subjects for material.Adopt alkaline-heating method to extract the genomic dna of corn embryosperm, concrete steps with embodiment 1.Carry out pcr amplification experiment with QK1.PCR reaction system is: PCR reaction system is: 10X Buffer 2 μ 1 (containing Mg2+), dNTP0.4 μ 1 (1OmM), each 2 μ 1 of nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:3 and sequence table SEQ ID NO:4 (5 μ Μ), genomic dna 1 μ 1 that corn to be measured extracts, taq enzyme 0.5 μ 1 (5U/ μ 1), reaction volume is 20 μ 1, drips a mineral oil and covers.PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 1Omin.
Pcr amplification product 6% Polyacrylamide Gel Electrophoresis, rapid silver staining dyes and observes Taking Pictures recording, 23 strains of the 234bp wherein isozygotying, 28 strains of the 274bp that isozygotys, 45 strains of heterozygosis banding pattern.
In the time of corn seeding, before earthing, by the corn kernel with pathogenic bacteria evenly cover 96 cut a little endosperm (get 1/20 corn kernel endosperm, the arbitrary position that can get endosperm only otherwise hurt maize) after F
2above seed.After inoculation, irrigate 1 time, after this keep sufficient soil moisture.The pathogenic bacteria using is provided by Agricultural University Of Anhui.
After morbidity, by field test, blade is blue or green withered or yellow withered, fruit ear reversal of the natural order of things, and stem foot deliquescing browning also rots, and thinks susceptible strain, and other is designated as disease-resistant plant.Field investigation result is carried out to record.
Be analyzed as follows: as shown in table 1 below, for 23 strains that have the 234bp that isozygotys in examination corn sample, 20 strains show as disease-resistant, have 3 strains susceptible; And susceptible without 26 strains in 28 materials of 234bp fragment, there are 2 strains disease-resistant; 45 strains of heterozygosis, 41 strains show as disease-resistant, and 4 strains are susceptible; Both consistence reach 90.6%.Therefore, sequence table SEQ ID NO:3 is and the mutually closely linked molecule marker of bacterial wilt resistance trait related locus to can be used for corn marker assisted selection with the QK1 primer pair of sequence table SEQ ID NO:4 composition.
Table 1 QK1 amplified production qualification result contrasts with field bacterial wilt inoculation test
Claims (9)
1. with the closely linked molecule marker of Corn Stalk Rot resistant gene, it is characterized in that, described molecule marker is nucleotide sequence shown in the nucleotide sequence shown in sequence table SEQ ID NO:1 or sequence table SEQ ID NO:2.
2. described in the claim 1 that increases, a primer pair for molecule marker, is characterized in that, the primer 1 of described primer pair is the nucleotide sequence shown in sequence table SEQ ID NO:3, and the primer 2 of described primer pair is the nucleotide sequence shown in SEQ ID NO:4.
3. a method that detects Corn Stalk Rot resistance, is characterized in that, described method comprises the steps:
(1) pcr amplification: the genome extracting using corn to be measured, as DNA cloning template, consists of amplimer to carrying out pcr amplification with nucleotide sequence shown in nucleotide sequence shown in sequence table SEQ ID NO:3 and sequence table SEQ ID NO:4;
(2) qualification of amplified production: if gained pcr amplification product is 234bp base fragment, treat that corn is resistance to bacterial wilt kind, if gained pcr amplification product is 274bp base fragment, corn to be measured is bacterial wilt susceptible variety.
4. a kind of method that detects Corn Stalk Rot resistance according to claim 3, is characterized in that, in described step (2), 234bp base fragment is the nucleotide sequence shown in sequence table SEQ ID NO:1; 274bp base fragment is nucleotide sequence shown in sequence table SEQ ID NO:2.
5. method according to claim 3, it is characterized in that, the genome method that corn to be measured in described step (1) extracts is alkaline-heating method, concrete steps are: get 1/20 corn kernel endosperm 0.2Mol/L sodium hydroxide 40ml 100 DEG C of water-baths 5 minutes, add again the 0.17Mol/Ltris-HCL water-bath 3 minutes of 60ml, 4 DEG C of refrigerators are cooling for subsequent use, obtain the genome that described corn to be measured extracts.
6. method according to claim 3, it is characterized in that, the reaction system of the pcr amplification in described step (1) is: containing the 10X Buffer2 μ 1 of Mg2+, the dNTP0.4 μ l of 1OmM, each 2 μ 1 of nucleotide sequence shown in nucleotide sequence shown in the sequence table SEQ ID NO:3 of 5 μ M and sequence table SEQ ID NO:4, genomic dna 1 μ 1 that described corn to be measured extracts, taq enzyme 0.5 μ 1 of 5U/ μ 1, all the other are ultrapure water, reaction volume is 20 μ 1, drips a mineral oil and covers; PCR response procedures is: 94 DEG C of 5min; 94 DEG C of 60s, 53.5 DEG C of 60s, 72 DEG C of 60s, 35 circulations; 72 DEG C of 1Omin.
7. the application of molecule marker according to claim 1 in corn breeding.
8. the application of primer pair according to claim 2 in corn breeding.
9. according to the application in corn breeding for detection of the method for Corn Stalk Rot resistance of any one in claim 3-6.
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Cited By (4)
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| CN107354215A (en) * | 2017-08-04 | 2017-11-17 | 中国农业大学 | A kind of corn molecule auxiliary breeding means |
| CN108004236A (en) * | 2017-12-28 | 2018-05-08 | 袁隆平农业高科技股份有限公司 | The disease-resistant molecular breeding method of corn stalk rot disease and its application |
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| CN108893550B (en) * | 2018-07-10 | 2021-08-10 | 北京市农林科学院 | Development and application of SNP molecular marker related to resistance of corn stalk rot |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018487A (en) * | 2015-08-06 | 2015-11-04 | 中国农业大学 | Molecular marker for major QTL of chromosome-3 ear row number of corn and application thereof |
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| CN105063201B (en) * | 2015-08-06 | 2018-02-23 | 中国农业大学 | The molecular labeling of corn Chromosome 9 tassel row number main effect QTL and its application |
| CN107354215A (en) * | 2017-08-04 | 2017-11-17 | 中国农业大学 | A kind of corn molecule auxiliary breeding means |
| CN108004236A (en) * | 2017-12-28 | 2018-05-08 | 袁隆平农业高科技股份有限公司 | The disease-resistant molecular breeding method of corn stalk rot disease and its application |
| CN108004236B (en) * | 2017-12-28 | 2020-11-06 | 袁隆平农业高科技股份有限公司 | Corn stalk rot disease-resistant molecular breeding method and application thereof |
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