CN112941217A - Melon downy mildew resistant InDel marker and application thereof - Google Patents
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Abstract
The invention specifically provides an InDel marker for resisting downy mildew of melons, InDel15 and InDel20 are used as molecular markers, the molecular markers and specific primers thereof obtained by the invention can be used for screening and identifying downy mildew resistant varieties at early stage, the accuracy of identification results is high, the determination results are reliable, the breeding period can be greatly shortened, the breeding efficiency is improved, and the invention has important theoretical and practical significance. The kit for screening the melon resources with the melon downy mildew resistance can clearly distinguish whether the melon materials are downy mildew resistant varieties or not through direct electrophoresis detection of PCR amplification products, is time-saving and labor-saving, and is not influenced by the environment.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and particularly relates to a molecular marker for resisting downy mildew of melons and the technical field of application of the molecular marker.
Background
The melon (Cucumis melo L.) is an important horticultural economic crop of the Cucumis genus of the Cucurbitaceae family, has sweet and fragrant fruits, is well liked by consumers and is popular in all countries of the world. Melon downy mildew is a fungal disease caused by the fungus downy mildew of cubeba (Pseudoperonospora cubensis (berk. etcut.) Rostov.). Under the condition of high temperature and high humidity, the disease is rapidly expanded, the yield and the quality of the melon are seriously influenced, and the disease is a destructive and worldwide disease. Chemical control of downy mildew not only increases planting cost and pollutes environment, but also causes pathogenic bacteria to generate drug resistance, so that cultivation of disease-resistant varieties is a fundamental way for cost reduction and efficiency improvement and environmental friendliness for control of melon downy mildew.
The conventional breeding method is used for breeding the disease-resistant materials, screening is carried out according to the phenotypic anti-infection characteristics of plants, the subjective judgment of a breeding person is taken as a standard, the instability is high, the selection efficiency is influenced due to insufficient inoculation or improper disease conditions, and the individual plants with the disease-resistant genes are difficult to screen accurately and quickly. By excavating molecular markers with disease resistance and applying the molecular markers to auxiliary screening breeding, phenotype selection is converted into genotype selection, and filial generations are screened in a seedling stage, so that the screening efficiency can be remarkably improved, and the breeding process is accelerated.
Disclosure of Invention
In order to overcome the defects of time and labor waste, easy environmental influence and the like in breeding disease-resistant materials by using the traditional method, an InDel molecular marker for resisting downy mildew of melons is not disclosed in the prior art. The invention provides an insertion-deletion marker molecular marker for resisting downy mildew of melons and application thereof, which can be used for auxiliary screening and breeding of downy mildew resistance and obviously quickens the speed and precision of new variety breeding.
The invention discloses an InDel molecular marker for resisting downy mildew of melons, which is InDel15 and InDel 20.
The nucleotide sequences of the primers for the Indel15 markers are as follows:
InDel 15F/InDel 15R:
5’-TCACAGCTCACAATCACAGGT-3’/5’-AGGGGAAGCTGGAGAAGACA-3’
the marker characteristic band of the primer amplified and the melon downy mildew resistant Indel15 is 251bp, and the nucleotide sequence of the marker characteristic band is shown as SeqID No. 3.
The Indel15 marked characteristic band amplified by the primer and resisting downy mildew of melon is 231bp, and the nucleotide sequence of the Indel15 marked characteristic band is shown as SeqID No. 4.
The nucleotide sequence of the Indel 20-labeled primers is as follows:
InDel 20F/InDel 20R:
5’-TGGGGTTGCTTGTGGATAGT-3’/5’-AGGCGAACACCTTTTGATTCA-3’
the Indel20 marked characteristic band amplified by the primer and resisting downy mildew of melon is 349bp, and the nucleotide sequence of the Indel20 marked characteristic band is shown as SeqID No. 7;
the Indel20 marked characteristic band amplified by the primer and resisting downy mildew of melon is 324bp, and the nucleotide sequence of the Indel20 marked characteristic band is shown as SeqID No. 8.
Further, the present invention provides a kit for screening for cucumis melo germplasm resources with resistance to cucumis melo downy mildew, comprising Indel-labeled primers;
the nucleotide sequence of the Indel-labeled primers is as follows:
the Indel marker characteristic band of the primer amplification and melon downy mildew resistance is 251bp or 231bp or 349bp or 324bp, and the nucleotide sequence is shown as SeqID No.3, SeqID No.4, SeqID No.7 and SeqID No. 8.
The kit may further comprise reagents required for performing a PCR reaction and/or electrophoresis.
Furthermore, the invention also provides a method for screening melon germplasm resources with melon downy mildew resistance, which adopts molecular markers InDel15 and InDel20 or the kit to carry out the following steps:
(1) extracting genome DNA of any tissue or organ of a muskmelon sample to be detected by using a CTAB method;
(2) taking the genome DNA of the sample to be detected obtained in the step (1) as a template, and carrying out PCR amplification on different materials by using a special molecular marker primer to obtain an amplification product;
(3) performing polyacrylamide gel electrophoresis on the PCR amplification product obtained in the step (2);
(4) detecting the result of gel electrophoresis by using a silver staining method, and observing the size of a PCR amplification product;
(5) through the identification of judging the size of the fragments, the amplified PCR product fragments are larger and are the muskmelon materials resisting downy mildew, and the amplified fragments are smaller and are the muskmelon materials resisting downy mildew.
Preferably, the reaction system for PCR amplification is:
2X
PCR SuperMix 10. mu.L, cDNA template 2.0. mu.L, Indel-labeled upstream and downstream primers (10. mu.M) each 0.5. mu. L, ddH2O7.0. mu.L, 20. mu.L in total.
Preferably, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing temperature InDel 15: 57 ℃/InDel 20: at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; final extension at 72 ℃ for 10min after 28 cycles
Preferably, the gel electrophoresis detection refers to separation by using 8% non-denaturing polyacrylamide gel at constant power of 120V/500mA for 90min, and finally silver staining for color development.
Meanwhile, the invention further provides a preparation method of the kit for screening melon germplasm resources for resisting downy mildew of melons, and reagents for assembling the kit comprise InDel molecular markers InDel15 and InDel20 amplification primers for resisting downy mildew of melons.
Meanwhile, reagents required for PCR reaction and/or electrophoresis are also included in the reagents of the assembly kit.
In the invention, the adopted downy mildew-resistant melon germplasm and the local variety 'yellow convex' of the susceptible Xinjiang melon are purchased and obtained in the market, common technicians can obtain the yellow convex in the market through public channels, and the culture conditions of the two melon germplasm resources can be obtained by adopting common reports in the field.
By implementing the specific invention content of the invention, the following beneficial effects can be achieved:
(1) the invention obtains the molecular marker of the melon downy mildew resistance InDel for the first time: InDel15 and InDel 20.
(2) The molecular marker and the specific primer thereof obtained by the invention can screen and identify the anti-downy mildew variety in the early stage, have high identification result accuracy and reliable determination result, can obviously shorten the breeding period and obviously improve the breeding efficiency, and have important theoretical and practical significance.
(3) By directly carrying out electrophoresis detection on the PCR amplification product, whether the melon material is a downy mildew resistant variety can be clearly distinguished, time and labor are saved, and the method is not influenced by the environment.
Drawings
FIG. 1 is a graph showing the results of InDel15 primer amplification.
Wherein 1: downy mildew resistant parent K7-6, 12: downy mildew susceptible parent yellow convex, 1-5: disease-resistant single plant, 6-10: infected individual, M: 2K DNAmarker.
FIG. 2 is a graph showing the amplification results of InDel20 primers.
Wherein 1: downy mildew susceptible parent xanthophyll, 2: the downy mildew resistant parent K7-6, 3-7: disease-resistant single plant, 8-12: infected individual, M: 2K DNAmarker.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples.
The materials of the invention are: liquid nitrogen, distilled water, double distilled water, chloroform: isoamyl alcohol (24:1), absolute ethyl alcohol, agarose, 2X
PCR Supermix, acrylamide stock, 5 XTBE, AP, TEMED.
The instruments used in the invention comprise the following materials:
centrifuge tube, steel ball, liquid nitrogen tank, centrifuge, constant temperature water bath, vortex oscillation instrument, ultramicro spectrophotometer, microblog, electrophoresis tank, electrophoresis apparatus, PCR instrument, and shaking table.
The reagents and materials can be purchased through public channels, and the equipment and instruments adopted in the process are common equipment in the field.
All materials, reagents and equipment selected for use in the present invention are well known in the art, but do not limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The first embodiment is as follows: muskmelon downy mildew resistant InDel molecular marker
The invention discloses an InDel molecular marker for resisting downy mildew of melons, which is InDel15 and InDel 20.
The nucleotide sequences of the primers for the Indel15 markers are as follows:
InDel 15F/InDel 15R:
5’-TCACAGCTCACAATCACAGGT-3’/5’-AGGGGAAGCTGGAGAAGACA-3’
the marked characteristic band of the melon downy mildew resistant Indel15 amplified by the primer is 251bp, and the nucleotide sequence of the marked characteristic band is shown as SeqID No. 3;
the marker characteristic band of the melon downy mildew resistant Indel15 amplified by the primer is 231bp, and the nucleotide sequence of the marker characteristic band is shown as SeqID No. 4.
The nucleotide sequence of the Indel 20-labeled primers is as follows:
InDel 20F/InDel 20R:
5’-TGGGGTTGCTTGTGGATAGT-3’/5’-AGGCGAACACCTTTTGATTCA-3’
the marker characteristic band of the melon downy mildew resistant Indel20 amplified by the primer is 349bp, and the nucleotide sequence of the marker characteristic band is shown as SeqID No. 7;
the Indel20 marked characteristic band of the melon downy mildew resistance amplified by the primer is 324bp, and the nucleotide sequence of the Indel20 marked characteristic band is shown as SeqID No. 8.
Example two: kit for screening melon resource with melon downy mildew resistance
Further, the present invention provides a kit for screening for cucumis melo germplasm resources with resistance to cucumis melo downy mildew, comprising Indel-labeled primers;
the nucleotide sequence of the Indel-labeled primers is as follows:
the primer amplified and melon downy mildew resistant Indel marker characteristic band is 251bp or 231bp or 349bp or 324bp, and the nucleotide sequences are shown as SeqID No.3, SeqID No.4, SeqID No.7 and SeqID No. 8.
The kit may further comprise reagents required for performing a PCR reaction and/or electrophoresis.
Example four: method for screening melon germplasm resources with melon downy mildew resistance
Furthermore, the invention also provides a method for screening melon germplasm resources with melon downy mildew resistance, which adopts the molecular markers InDel15 and InDel20 or the kit to carry out the following steps:
(1) extracting genome DNA of any tissue or organ of a muskmelon sample to be detected by using a CTAB method;
(2) taking the genome DNA of the sample to be detected obtained in the step (1) as a template, and carrying out PCR amplification on different materials by using a special molecular marker primer to obtain an amplification product;
(3) performing polyacrylamide gel electrophoresis on the PCR amplification product obtained in the step (2);
(4) detecting the result of gel electrophoresis by using a silver staining method, and observing the size of a PCR amplification product;
(5) through the identification of judging the size of the fragments, the amplified PCR product fragments are larger and are the muskmelon materials resisting downy mildew, and the amplified fragments are smaller and are the muskmelon materials resisting downy mildew.
Preferably, the reaction system for PCR amplification is:
Preferably, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing temperature InDel 15: 57 ℃/InDel 20: at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; final extension at 72 ℃ for 10min after 28 cycles
Preferably, the gel electrophoresis detection refers to separation by using 8% non-denaturing polyacrylamide gel at constant power of 120V/500mA for 90min, and finally silver staining for color development.
Example five: preparation method of kit for screening melon germplasm resources with downy mildew resistance of melons
Meanwhile, the invention further provides a preparation method of the kit for screening melon germplasm resources for resisting downy mildew of melons, and reagents of the assembled kit comprise amplification primers of InDel molecular markers InDel15 and InDel20 for resisting downy mildew of melons. The reagents of the assembly kit also include reagents required for PCR reaction and/or electrophoresis.
Example six: genotype verification analysis of F2-generation segregation population material
The results of PCR amplification of 181 individuals of the parental, F2 isolate population were determined by the markers, kits and screening methods provided in the fifth example, which corresponds to the above example:
after the primer InDel15 is subjected to PCR amplification, among 100 single plants with disease-resistant phenotype identification results, the genotype detection results of 91 single plants are all shown as disease-resistant banding types, and the detection results of 9 disease-resistant plants are shown to be inconsistent with the phenotype results, so that the disease-resistant single plants are shown as cross-over plants; among 81 individuals with the disease-sensitive phenotype identification results, 79 individuals have the disease-sensitive genotype identification results, and 2 individuals with the disease-sensitive phenotype detection results do not accord with the phenotype results, so that the disease-sensitive individuals are the cross-over plants; thus, there were 11 crossover individuals in the 181 individuals of the F2 population. The results of some of the samples are shown in FIG. 1.
After the primer InDel20 is subjected to PCR amplification, among 100 single plants with disease-resistant phenotype identification results, the genotype detection results of 97 single plants are all shown as disease-resistant banding types, and the detection results of 3 disease-resistant plants are shown to be inconsistent with the phenotype results, which indicates that the disease-resistant single plants are crossover plants; among 81 individuals with disease-sensitive phenotype identification results, 80 individuals have disease-sensitive genotype identification results, and 1 individual-susceptible-phenotype detection result is inconsistent with the phenotype result, which indicates that the individual-susceptible strain is a cross strain; thus, there were 4 crossover individuals in the 181 individuals of the F2 population. The results of some of the samples are shown in FIG. 2.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Sequence listing
<110> Hami melon research center of Xinjiang academy of agricultural sciences
<120> melon downy mildew-resistant InDel marker and application thereof
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<213> melon (Cucumis melo L.)
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agtggtggtt tagggacaga acatatagtg tacggaatca aagtggggaa agtgatgggg 120
aacttgagaa ttgagaacga aaaagagaga cccaattcca ctatactctg taatctacaa 180
tctatcattc ttttgtttat tattattatt cacaatcaca tccaccaatt ttgtcttctc 240
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<210> 4
<211> 231
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<213> melon (Cucumis melo L.)
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tcacagctca caatcacagg taaagtagag agagagagtg agtggtggtt tagggacaga 60
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aaaagagaga cccaattcca ctatactctg taatctacaa tctatcattc ttttgtttat 180
tattattatt cacaatcaca tccaccaatt ttgtcttctc cagcttcccc t 231
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caaaacaaaa gtcgtctttt ataaatagtt taatttattt tgaagttaca aacgatggat 180
aatgcaaaaa ttgattaact ttcataccta tatcttgttt gagacaaaag aaaaatttta 240
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acctatatct tgtttgagac aaaagaaaaa ttttatatta actaacctta agttatcgag 240
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Claims (10)
1. An Indel marker for resisting downy mildew of melons is characterized in that: contains Indel15 markers, and the nucleotide sequences of primers of the Indel markers are shown as SeqID No.1 and SeqID No. 2;
the marked characteristic band of the melon downy mildew resistance marker Indel15 amplified by the primer is 251bp, and the nucleotide sequence of the marked characteristic band is shown as SeqID No. 3;
the marker signature band of the melon downy mildew resistance marker Indel15 amplified by the primer is 231bp, and the nucleotide sequence of the marker signature band is shown as SeqID No. 4.
2. An Indel marker for resisting downy mildew of melons is characterized in that: contains Indel20 markers, and the nucleotide sequences of primers of the Indel markers are shown as SeqID No.3 and SeqID No. 4;
the marker characteristic band of the melon downy mildew resistance marker Indel20 amplified by the primer is 349bp, and the nucleotide sequence of the marker is shown as SeqID No. 7;
the marker characteristic band of the melon downy mildew resistance marker Indel20 amplified by the primer is 324bp, and the nucleotide sequence of the marker is shown as SeqID No. 8.
3. A kit for screening a cucumis melo mass source for resistance to cucumis melo downy mildew, comprising a primer for the Indel marker of claim 1 or claim 2;
the marker characteristic band of the melon downy mildew resistant Indel amplified by the primer is 251bp or 231bp or 349bp or 324bp, and the nucleotide sequence of the marker characteristic band is shown as SeqID No.3, SeqID No.4, SeqID No.7 and SeqID No. 8.
4. The kit of claim 3, further comprising reagents required for performing a PCR reaction and/or electrophoresis.
5. Method for screening melon germplasm resources for resistance to melon downy mildew, characterized in that the Indel marker according to claim 1 and claim 2 or the kit according to any of claims 3 to 4 is used for the following steps:
(1) extracting genome DNA of any tissue or organ of a muskmelon sample to be detected by using a CTAB method;
(2) taking the genome DNA of the sample to be detected obtained in the step (1) as a template, and carrying out PCR amplification on different materials by using a special molecular marker primer to obtain an amplification product;
(3) performing polyacrylamide gel electrophoresis on the PCR amplification product obtained in the step (2);
(4) detecting the result of gel electrophoresis by using a silver staining method, and observing the size of a PCR amplification product;
(5) through the identification of judging the size of the fragments, the amplified PCR product fragments are larger and are the muskmelon materials resisting downy mildew, and the amplified fragments are smaller and are the muskmelon materials resisting downy mildew.
6. The method of claim 5, wherein the PCR amplification reaction system is:
2X
PCR SuperMix 10. mu.L, cDNA template 2.0. mu.L, Indel-labeled upstream and downstream primers (10. mu.M) each 0.5. mu. L, ddH2O7.0. mu.L, 20. mu.L in total.
7. A method as claimed in any one of claims 5 or 6, wherein the PCR amplification reaction sequence is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing temperature InDel 15: 57 ℃/InDel 20: at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; the final extension was 10min at 72 ℃ after 28 cycles.
8. A method as claimed in any one of claims 5 to 7, wherein said gel electrophoresis assay is 8% native polyacrylamide gel separation at 120V/500mA constant power electrophoresis for 90min, and finally silver staining for color development.
9. A method for preparing a kit for screening melon germplasm resources resistant to downy mildew of melons, wherein a reagent for assembling the kit comprises a marker primer marked by Indel as claimed in claim 1 or claim 2.
10. The method of claim 9, wherein the reagents of the kit further comprise reagents required for PCR reaction and/or electrophoresis.
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| CN105734057A (en) * | 2016-04-29 | 2016-07-06 | 新疆农业科学院哈密瓜研究中心 | SSR mark linked with pseudoperonospora cubensis resistance main effect QTL and application of SSR mark |
| US20180146633A1 (en) * | 2014-06-06 | 2018-05-31 | Nunhems B.V. | MELON PLANTS WITH A DOMINANT MELON YELLOWING ASSOCIATED VIRUS (MYaV) RESISTANCE GENE |
| CN109601367A (en) * | 2018-12-06 | 2019-04-12 | 南京农业大学 | The breeding method of downy mildew resistance cucumber interspecific hybrid new varieties and application |
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