CN116287414A - Molecular marker for detecting corn stem rot resistance gene ZmCCT and application thereof - Google Patents
Molecular marker for detecting corn stem rot resistance gene ZmCCT and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker for detecting a corn stem rot resistance gene ZmCCT and application thereof, wherein the linkage position of the molecular marker is the insertion or deletion of 94435769bp-94440892bp of a corn reference genome Maize B73genome sequences AGPv No. 10 chromosome. The invention utilizes the specificity molecular marker linked with the corn stem rot resistance and combines with KASP detection technology, agarose gel electrophoresis is not needed, and the method can rapidly, accurately, efficiently and high-throughput identify the corn stem rot resistance material and accelerate the breeding process of new corn stem rot resistance varieties.
Description
Technical Field
The invention belongs to the field of agricultural molecular biology, and particularly relates to a molecular marker for detecting a corn stem rot resistance gene ZmCCT and application thereof.
Background
Corn stem rot is a disease caused by various pathogenic bacteria and seriously affects the production of corn. There are up to 20 fungi or bacteria worldwide that can infect corn to induce stem rot, and are classified into 5 types including pythium, gibberella, fusarium, anthrax, and bacteria. Corn stalk rot mainly comprises humic acid bacteria and fusarium, wherein fusarium is the main pathogenic bacteria of the stalk rot, and fusarium is the most serious hazard of fusarium graminearum, fusarium layering and fusarium verticillatum. Pathogenic bacteria mainly infect the root system and the stem base of the corn, cause the stem base to rot, influence the normal growth of the corn, and finally cause the yield to be reduced. The stem rot disease is rapid in development and serious in hazard, once the corn is infected, the yield of light people is reduced by 10% -30%, and the yield can reach more than 50% when serious. At present, the stem rot is prevented and treated by commonly used chemical agents in production, and a large amount of pesticides not only pollute the environment, but also cause the drug resistance of pathogenic bacteria, thereby increasing the difficulty in preventing and treating the stem rot.
Compared with chemical agent control, the cultivation of corn stem rot resistant varieties has the characteristics of safety, environmental protection and the like, and has outstanding advantages in stem rot control. In recent years, related technologies clone or locate a plurality of genes related to stem rot resistance, and provide a theoretical basis for breeding of corn stem rot resistant varieties. The identification of the resistance of the corn stem rot requires artificial inoculation of germs, is easily influenced by environment, and can deviate measured data. At present, the molecular markers for breeding the corn stem rot resistant varieties are fewer, complicated gel electrophoresis detection is needed for the molecular markers, the automation degree is low, the flux is small, and the breeding process of the corn stem rot resistant varieties is greatly limited. Therefore, the development of the corn stem rot resistance linkage KASP molecular marker can rapidly screen disease-resistant materials with high flux, quicken the breeding process of new varieties and has important significance for reducing yield loss caused by corn stem rot.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a molecular marker for detecting the corn stem rot resistance gene ZmCCT.
The invention also provides a primer group for detecting the molecular marker.
The invention also provides a kit.
The invention also provides a gene chip.
The invention also provides application of the molecular marker, the primer group, the kit and/or the gene chip.
The invention also provides a detection method of the molecular marker.
According to the embodiment of the first aspect of the invention, the molecular marker for detecting the corn stem rot resistance gene ZmCCT is provided, and the linkage position of the molecular marker is the insertion or deletion of 94435769bp-94440892bp of chromosome 10 of a corn reference genome Maize B73genome sequences AGPv.
In some embodiments of the invention, the sequence of the molecular marker is shown in SEQ ID NO. 4.
According to a second aspect of the present invention, a Primer set for amplifying the above molecular markers comprises a specific Primer and a universal Primer, wherein the specific Primer sequence comprises Primer X and Primer Y.
In some embodiments of the invention, the specific primer comprises a nucleotide sequence as set forth in SEQ ID NO.1 and SEQ ID NO. 2.
In some embodiments of the invention, the molecular markers further comprise a universal primer sequence having a nucleotide sequence as shown in SEQ ID NO. 3.
In some embodiments of the invention, the specific primer is linked to FAM and HEX fluorescent linker sequences, respectively.
According to an embodiment of the third aspect of the present invention, there is provided a kit comprising the above primer set.
According to a fourth aspect of the present invention, there is provided a gene chip comprising the above primer set.
According to a fifth aspect of the present invention, any one of the following uses of the above molecular markers, primer sets, kits or gene chips:
(1) The application in the genotyping of the corn stem rot resistance gene ZmCCT;
(2) The application in detecting corn stem rot resistance gene ZmCCT;
(3) Application in identifying and screening corns with stem rot resistance;
(4) Application in corn molecular marker assisted breeding;
(5) Application in corn breeding;
(6) The application in preparing products for corn breeding.
According to a sixth aspect of the present invention, a method for detecting corn stalk rot resistance using the above molecular markers, the method comprising the steps of:
s1, extracting genome DNA from corn;
s2, detecting the molecular marker of the genome DNA extracted in the step S1, and judging the stem rot resistance of the corn to be detected according to a detection result.
In some embodiments of the invention, only the base corresponding to Primer X is detected, and it is determined that the tested corn material does not carry a zmct resistance gene and does not have stem rot resistance; if only the base corresponding to the Primer Y is detected, judging that the tested corn material carries ZmCCT resistance genes and has stem rot resistance; if bases corresponding to the Primer X and the Primer Y are detected at the same time, judging that the tested corn material carries the ZmCCT resistance gene, is a heterozygous genotype and has stem rot resistance.
In some embodiments of the invention, preferably, in step S1, genomic DNA is extracted from corn using a simplified CTAB process (cetyl trimethylammonium bromide process).
In some embodiments of the invention, preferably, in step S2, the molecular markers are detected using the KASP (competitive allele-specific PCR) technique.
A method of maize breeding comprising the steps of: by using the genotype detection method, a sample with stem rot resistance is selected for subsequent breeding.
According to some embodiments of the invention, at least the following benefits are provided: the invention utilizes the specificity molecular marker linked with the corn stem rot resistance and combines with KASP detection technology, agarose gel electrophoresis is not needed, and the method can rapidly, accurately, efficiently and high-throughput identify the corn stem rot resistance material and accelerate the breeding process of new corn stem rot resistance varieties.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a molecular marker development flow chart in example 1 of the present invention;
FIG. 2 shows the typing results of the marker Zm900002_K02 in the donor and acceptor samples in example 1 of the present invention;
FIG. 3 shows the typing results of the marker Zm900002_K02 in example 1 of the present invention in 10 samples.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The embodiment of the invention comprises the following steps: molecular marker for detecting corn stem rot resistance gene ZmCCT
The design process of the molecular marker is shown in figure 1, and related researches show that the ZmCCT gene has fusarium graminearum stem rot resistance and can be used for improving corn stem rot resistant varieties. Specific mutation sites linked with the target genes are searched by comparing the re-sequencing data of the target genes and the upstream and downstream sequences of the target genes in the susceptible varieties, flanking sequences of the specific mutation sites are extracted, and the markers are screened and tested by designing and synthesizing primer sequences of the markers, wherein the specific mutation sites are as follows:
1 primer design
Specific mutation sites linked with a target gene are searched by comparing the resequencing data of ZmCCT genes and upstream and downstream sequences thereof in the influenza material, and fragment insertion/deletion mutation is found at the position of chromosome 10 94435769-94440892 in a reference genome Maize B73genome sequences AGPv, wherein the position is positioned upstream of the genes, and a BatchPrimer3 primer design website is utilized for primer design of the position. The design sequence of the molecular marker Zm900002_K02 is shown as SEQ ID NO.1, the primer set designed according to the molecular marker consists of 3 primers, as shown in Table 1, wherein the 5' ends of the 2 specific primers are respectively connected with FAM and HEX fluorescent linker sequences, and 1 universal primer. The primer is entrusted to Shanghai biochemical synthesis.
Molecular marker sequence (SEQ ID NO. 4):
CGAACCACAAGCATGACGACCCAATGGCAATGGAGGTCGAGATAGATGGATA TCTCCGTA [ GCTCACTACAGGAAAACGACCAGTTTCCGACGGCCTACACTGTTTCC GACG … … GGCCGTCGGAAACTGGTCATTTTCCTGTAGTG/- ] GCTACTAGCTATAGCT TTGTTAAAAGGCAGCAGCAAAATTCGAAGCTGGGGCCAGCGGCC. The omitted parts are identical to the sequence in chromosome 10 94435801-94440841 in Maize B73genome sequences AGPv.
By using the KASP reaction principle, the genotyping of the sample can be performed with high throughput. If the PCR product only detects a fluorescence signal corresponding to the Primer X, the base of the detection site is C, and the test material does not carry a ZmCCT stem rot resistance gene; if only the fluorescence signal corresponding to the Primer Y is detected, the base of the detection site is G, and the test material carries a ZmCCT stem rot resistance gene; if the fluorescence signals corresponding to the Primer X and the Primer Y are detected at the same time, the base of the detection site is C to G, and the test material is heterozygous genotype and carries ZmCCT stem rot resistance genes.
TABLE 1 marker information
2 sample detection
DNA extraction: extracting genome DNA from corn leaves by adopting a simplified CTAB method, comprising the following steps:
(1) Taking about 30mg of blades to 1.3mL of a 96-well plate, placing the blades in a freeze dryer, and vacuumizing for 12 hours or more;
(2) After vacuumizing, adding two steel balls into each hole by using a bead divider, covering a silica gel film, grinding for 1min in a high-flux grinding instrument, immediately separating in a deep-hole plate centrifuge, and centrifuging the ground tissue to the bottom of the hole;
(3) Adding 700 mu L of CTAB extracting solution into each hole by using a pipetting workstation TECAN, shaking and uniformly mixing, placing into a 65 ℃ water bath kettle for warm bath for about 1-1.5h, taking 1.3mL of 96-well plates on a vortex oscillator for shaking for several times every 20 min;
(4) Taking out 1.3mL 96-well plate after the warm bath is finished, placing the 96-well plate into a deep-well plate refrigerated centrifuge, and centrifuging at 4000rpm for 10min;
(5) Transferring 380 mu L of supernatant in each well to a new 1.3mL 96-well plate by using a pipetting workstation TECAN, adding equal volume chloroform, mixing uniformly upside down, standing for 2min, placing in a deep-hole plate refrigerated centrifuge, centrifuging at 4000rpm for 10min;
(6) After centrifugation, 250. Mu.L of supernatant is extracted by a pipetting workstation TECAN to 0.8mL of 96-well plate with 250. Mu.L of isopropanol added in advance, and the mixture is uniformly mixed by vortex oscillation and placed in a refrigerator at the temperature of minus 20 ℃ for precipitation for 1 hour or more;
(7) Taking out 0.8mL of the 96-well plate, placing the 96-well plate in a deep-hole plate refrigerated centrifuge, centrifuging at 4000rpm for 15min;
(8) Discarding the supernatant, adding 250 μL of 70% ethanol into each well by using a pipetting workstation TECAN, oscillating for several times on a vortex oscillator, centrifuging for 15min at 5000 rpm;
(9) Discarding the supernatant, and placing in a 65 ℃ oven for 30min to dry;
(10) 200. Mu.L of sterilized ultrapure water was added to each well, and the mixture was left at room temperature overnight for dissolution.
KASP reaction test: the KASP reaction test was performed on a Douglas Arraytape genotyping platform, the reaction system is shown in table 2, from which it can be seen that the amplification system used for the PCR amplification reaction was 0.8 μl: after 20ng-50ng of the sample DNA was dried, 0.0013. Mu.L of each of 100. Mu.M of the two specific primers, 0.0033. Mu.L of 100. Mu.M of the universal primer, 0.3945. Mu.L of 2 XSKASP Master Mix, and 0.3996. Mu.L of ultrapure water were added. The PCR amplification is completed in a water bath thermal cycler, and the TouchDown PCR reaction conditions are as follows: pre-denaturation at 94℃for 15min; the first step of amplification reaction, denaturation at 94 ℃ for 20s, annealing at 65-57 ℃ and extension for 60s,10 cycles, wherein the annealing and extension temperature in each cycle is reduced by 0.8 ℃; the second amplification step was performed by denaturation at 94℃for 20s, annealing at 57℃and extension for 60s for 30 cycles. After the reaction is completed, the fluorescent data of KASP reaction products are read by using an Arrayape scanning system, and the result of fluorescent scanning is automatically converted into a pattern.
TABLE 2KASP detection reaction System
| Final concentration | Volume of | |
| 100μM Primer C | 0.42μM | 0.0033μL |
| 100μM Primer X | 0.17μM | 0.0013μL |
| 100μM Primer Y | 0.17μM | 0.0013μL |
| 2×KASP Master Mix | 1× | 0.3945μL |
| Ultrapure water | 0.3996μL | |
| DNA (drying) | 20ng-50ng | |
| Total volume of | 0.8μL |
3 mark type data
According to the above assay, the KASP reaction was verified with the marker Zm900002_k02 on stem rot improvement donor zier 319 and stem rot improvement acceptor DH840 of known genotypes, 5 replicates per sample.
The detection result is shown in fig. 2, and it can be seen from the graph that the detection result of stem rot improvement donor zizania 319 at the target site is base G and carries ZmCCT stem rot resistance gene; the detection result of the stem rot improvement receptor DH840 at the target site is a base C, and the ZmCCT stem rot resistance gene is not carried.
4 specificity and practicality assays
To test the specificity and practicality of the marker Zm900002_k02 of the invention, 10 samples of known anti-susceptibility phenotype were identified, 3 replicates per sample, according to the test method described above.
The marking type result is shown in figure 3, and the marking Zm900002_K02 detects 8 parts of C homozygous genotype sample, and does not carry ZmCCT stem rot resistance gene; and 2G homozygous genotype samples are detected, the genotype samples carry ZmCCT stem rot resistance genes, the typing results completely meet the expectations, and the typing graph is shown in figure 2. The marker Zm900002_K02 has high specificity when detecting the corn ZmCCT stem rot resistance gene, can accurately and efficiently identify fusarium graminearum stem rot resistance materials, and accelerates the breeding process of corn stem rot resistance varieties.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The molecular marker for detecting the corn stem rot resistance gene ZmCCT is characterized in that the linkage position of the molecular marker is the insertion or deletion of 94435769bp-94440892bp of a corn reference genome Maize B73genome sequences AGPv4 chromosome 10.
2. A primer set for amplifying the molecular marker according to claim 1.
3. The primer set of claim 2, wherein the primer set comprises a specific primer comprising the nucleotide sequences set forth in SEQ ID No.1 and SEQ ID No. 2.
4. The primer set of claim 3 wherein the specific primer is linked to FAM and HEX fluorescent linker sequences, respectively.
5. The primer set of claim 2, wherein the primer set further comprises a universal primer having a nucleotide sequence set forth in SEQ ID No. 3.
6. A kit comprising the primer set of any one of claims 2-5.
7. A gene chip comprising the primer set according to any one of claims 2 to 5.
8. The molecular marker of claim 1, the primer set of any one of claims 2 to 5, the kit of claim 6 or any one of the following applications of the gene chip of claim 7:
(1) The application in the genotyping of the corn stem rot resistance gene ZmCCT;
(2) The application in detecting corn stem rot resistance gene ZmCCT;
(3) Application in identifying and screening corns with stem rot resistance;
(4) Application in corn molecular marker assisted breeding;
(5) Application in corn breeding;
(6) The application in preparing products for corn breeding.
9. A method for detecting corn stalk rot resistance using the molecular marker according to claim 1, comprising the steps of:
s1, extracting genome DNA from corn;
s2, carrying out polymorphism detection on the genome DNA extracted in the step S1, and judging stem rot resistance of the corn to be detected according to detection results.
10. A method of maize breeding comprising the steps of: the method of claim 9, selecting a sample with stem rot resistance for subsequent breeding.
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| CN202310346955.1A CN116287414A (en) | 2023-04-03 | 2023-04-03 | Molecular marker for detecting corn stem rot resistance gene ZmCCT and application thereof |
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| CN202310346955.1A CN116287414A (en) | 2023-04-03 | 2023-04-03 | Molecular marker for detecting corn stem rot resistance gene ZmCCT and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107354215A (en) * | 2017-08-04 | 2017-11-17 | 中国农业大学 | A kind of corn molecule auxiliary breeding means |
| CN109504749A (en) * | 2018-12-07 | 2019-03-22 | 袁隆平农业高科技股份有限公司 | The KASP detection primer of transgenic corns L239 and its filial generation homozygote and heterozygote |
| CN111295447A (en) * | 2017-06-15 | 2020-06-16 | 先正达农作物保护股份公司 | Maize elite event MZIR098 |
| CN115552038A (en) * | 2019-12-20 | 2022-12-30 | 科沃施种子欧洲股份两合公司 | Enhancement of maize disease resistance to northern leaf blight by QTL on chromosome 4 |
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- 2023-04-03 CN CN202310346955.1A patent/CN116287414A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111295447A (en) * | 2017-06-15 | 2020-06-16 | 先正达农作物保护股份公司 | Maize elite event MZIR098 |
| CN107354215A (en) * | 2017-08-04 | 2017-11-17 | 中国农业大学 | A kind of corn molecule auxiliary breeding means |
| CN109504749A (en) * | 2018-12-07 | 2019-03-22 | 袁隆平农业高科技股份有限公司 | The KASP detection primer of transgenic corns L239 and its filial generation homozygote and heterozygote |
| CN115552038A (en) * | 2019-12-20 | 2022-12-30 | 科沃施种子欧洲股份两合公司 | Enhancement of maize disease resistance to northern leaf blight by QTL on chromosome 4 |
Non-Patent Citations (1)
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| "NCBI_Release102_Chr10_94435769..94440892", pages 1, Retrieved from the Internet <URL:maizegdb.org> * |
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