CN110760602A - SSR primer and method for identifying watermelon anthracnose resistance - Google Patents
SSR primer and method for identifying watermelon anthracnose resistance Download PDFInfo
- Publication number
- CN110760602A CN110760602A CN201810826669.4A CN201810826669A CN110760602A CN 110760602 A CN110760602 A CN 110760602A CN 201810826669 A CN201810826669 A CN 201810826669A CN 110760602 A CN110760602 A CN 110760602A
- Authority
- CN
- China
- Prior art keywords
- watermelon
- identifying
- sample
- anthracnose
- anthracnose resistance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an SSR primer for identifying resistance of watermelon anthracnose, which relates to the breeding and application technology of agricultural melons and vegetables, and comprises a forward primer 5'-AGAGGAATGTGGTAATGAATAAACATCT-3' and a reverse primer 5'-AATTGGCCCAAATATCCATATGAC-3'; the invention discloses a method for identifying the resistance of watermelon anthracnose by adopting an SSR primer for identifying the resistance of watermelon anthracnose, which is quick and efficient: the detection can be carried out only after the seeds of the watermelon materials germinate for 3-4 days, the detection process only needs 1-2 days and is not limited by the environment, and the work of identifying the resistance of the watermelon anthracnose can be completed in the early stage of watermelon plants; the accuracy is high, and the error caused by the environment influence of a pathogen identification method of artificial inoculation is avoided; and the operation is simple and the cost is low.
Description
Technical Field
The invention relates to the technical field of agricultural melon and vegetable breeding and application, in particular to an SSR primer and an SSR method for identifying watermelon anthracnose resistance.
Background
Watermelon (watermelon species, tomato.) Matsum Nakai belongs to the genus Citrullus in the family Cucurbitaceae, has high nutritive value, contains much water in fruits, tastes sweet and cool, is well liked by consumers, has high economic value and plays an important role in agricultural production. With the rapid increase of watermelon cultivation area, especially facility cultivation area, various diseases are increasingly aggravated. Especially watermelon anthracnose, has become a prominent problem in watermelon production.
Watermelon Anthracnose (Anthracnose) is a fungal disease caused by Colletotrichum cucurbitacearum (Pass.) Ell.etHalst, which occurs in many countries, and is particularly serious in high-temperature multi-wetland areas, and the disease becomes one of the main factors influencing the yield and quality of watermelons. Chemical control is the most common method for controlling anthracnose at present, but long-term use of chemical pesticides can cause environmental pollution and residual toxicity accumulation; beneficial microorganisms are killed, the balance of microbial populations in plants is damaged, and the occurrence of diseases is accelerated; pathogenic bacteria resistance increase and the like. By screening the anti-anthracnose watermelon germplasm resources, the method for cultivating the new disease-resistant watermelon variety is the most effective method for preventing and treating the disease.
The traditional breeding makes great contribution to the breeding of new watermelon varieties, but the following defects exist in the conventional breeding work of the watermelon for resisting anthracnose: large workload, long breeding period, less resistant germplasm resources, slow breeding process, great influence by environmental factors, unsatisfactory breeding effect and the like. If the anti-anthracnose and non-anti-anthracnose watermelon materials can be identified and screened in the early stage of watermelon plants, the breeding workload can be greatly reduced, the breeding process is accelerated, the breeding period is shortened, and a better breeding effect is brought.
Therefore, a scheme for identifying and screening the anti-anthracnose and non-anti-anthracnose watermelon materials in early watermelon plants is needed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the SSR primer and the method for identifying the resistance of watermelon anthracnose, and the method can be used for identifying and screening the anti-anthracnose and non-anti-anthracnose watermelon materials in early stages of watermelon plants.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: an SSR primer for identifying watermelon anthracnose resistance: the SSR primer has a forward primer of 5'-AGAGGAATGTGGTAATGAATAAACATCT-3' and a nucleotide sequence shown as SEQ ID NO.1 in a sequence table; the reverse primer is 5'-AATTGGCCCAAATATCCATATGAC-3', and the nucleotide sequence is shown as SEQ ID NO.2 in the sequence table.
The invention also discloses a method for identifying the resistance of the watermelon anthracnose by adopting the SSR primer for identifying the resistance of the watermelon anthracnose, which comprises the following steps:
extracting DNA of a watermelon sample to be detected;
carrying out PCR amplification on the extracted DNA of the watermelon sample to be detected by using a fluorescence-labeled SSR primer;
and performing capillary electrophoresis fluorescence detection on the PCR amplification product, and judging whether the watermelon has anthracnose resistance according to the detection result.
On the basis of the technical scheme, the method for extracting the DNA of the watermelon sample to be detected specifically comprises the following steps:
placing a watermelon sample to be detected in a centrifuge tube, adding liquid nitrogen, and smashing the sample to be detected by using a tissue triturator;
adding cetyl trimethyl ammonium bromide CTAB preheating buffer solution into a centrifuge tube, carrying out water bath, adding chloroform-isoamylol, uniformly mixing and centrifuging;
taking the supernatant, adding the supernatant into a centrifugal tube filled with isopropanol, uniformly mixing and centrifuging;
discarding the supernatant, washing the precipitate with 70% ethanol solution, drying, and adding double distilled water.
On the basis of the technical scheme, before a sample to be detected is smashed by a tissue triturator, a diethyl dithiocarbamate DIECA crystal is scattered on the sample to be detected.
On the basis of the technical scheme, before water bath, activated carbon is added into the centrifuge tube.
On the basis of the technical scheme, the PCR amplification of the DNA of the extracted watermelon sample to be detected by using the SSR primer comprises the following steps:
adding deoxyribonucleoside triphosphate, a fluorescence-labeled forward primer and a reverse primer, TaqDNA polymerase and a PCR buffer solution containing Mg2+ into a PCR tube, uniformly mixing the DNA of a watermelon sample to be detected and double distilled water, and placing the mixture on a PCR instrument for PCR amplification.
On the basis of the technical scheme, the reaction process of PCR amplification comprises the following steps: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s for 34 cycles; extending for 10min at 72 ℃, and storing for later use at 4 ℃.
On the basis of the technical scheme, the method for carrying out capillary electrophoresis fluorescence detection on the PCR amplification product and judging whether the watermelon has anthracnose resistance according to the capillary electrophoresis fluorescence detection result specifically comprises the following steps:
diluting PCR amplification products of 6-FAM fluorescent marker and HEX fluorescent marker by 30 times with ultrapure water respectively, and diluting PCR amplification products of TAMRA fluorescent marker and ROX fluorescent marker by 10 times with ultrapure water respectively;
respectively taking the diluted solutions of the 4PCR amplification products with the same volume, mixing to prepare a mixed solution, adding the mixed solution into a deep hole plate special for a DNA analyzer, and respectively adding an LIZ500 molecular weight internal standard and deionized formamide into each hole in the deep hole plate special for the DNA analyzer;
denaturing at 95 ℃ on a PCR analyzer, taking out and cooling, instantly centrifuging, and placing on a DNA analyzer for capillary electrophoresis detection;
using fragment analysis software of a DNA analyzer to analyze images, and making capillary electrophoresis data into an electrophoresis detection map; and judging whether the watermelon has the anthracnose resistance or not according to the electrophoresis detection map.
On the basis of the technical scheme, the watermelon sample to be detected comprises a disease-resistant parent, a disease-susceptible parent and filial generations of the disease-resistant parent and the disease-susceptible parent.
Compared with the prior art, the invention has the advantages that:
(1) the invention provides an SSR primer for identifying the resistance of watermelon anthracnose, which is based on an SSR marking technology, and DNA is not influenced by the environment, so that errors caused by the influence of the environment by a pathogen identification method of artificial inoculation are avoided; the detection result has high accuracy, and the detection result is compared with the purity contrast of the inoculation identification, and the results of the detection result and the purity contrast are highly consistent.
(2) The invention provides a method for identifying the resistance of watermelon anthracnose by adopting an SSR primer for identifying the resistance of watermelon anthracnose, which has the following advantages:
a. fast and efficient: the method can detect the watermelon anthracnose resistance only after the seeds of the watermelon materials germinate for 3-4 days, the detection process only needs 1-2 days and is not limited by the environment, and the method can finish the work of identifying the resistance of the watermelon anthracnose in the early stage of watermelon plants.
b. The accuracy is high: the method is based on SSR marking technology, and DNA is not influenced by environment, so that errors caused by environment influence in a pathogen identification method of artificial inoculation are avoided; the detection result is compared with the purity control of inoculation identification, and the results of the detection result and the purity control are highly consistent.
c. The operation is simple: the equipment and reagents required by the method are owned by a conventional laboratory, the programmed operation is simple and easy to implement, and no special theoretical basis is needed for operators.
d. The cost is low: the method saves various equipments and management required for planting watermelon, and saves a large amount of manpower, land and material resources.
Drawings
FIG. 1 is an electrophoresis detection spectrum of an E.coli material sample of the method for identifying the resistance of watermelon anthracnose by using the SSR primers for identifying the resistance of watermelon anthracnose in the embodiment of the invention; samples of the watermelon material with the male mark of HR 31;
FIG. 2 is an electrophoresis detection spectrum of a sample of a male parent PI189225 watermelon material in the method for identifying resistance to watermelon anthracnose by using an SSR primer for identifying resistance to watermelon anthracnose in the embodiment of the invention; (ii) a
FIG. 3 is an electrophoresis detection spectrum of a sample 1 randomly extracted from an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention;
FIG. 4 is an electrophoresis detection spectrum of a sample 2 randomly extracted from an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention;
FIG. 5 is an electrophoresis detection spectrum of a sample 3 randomly extracted from an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention;
FIG. 6 is an electrophoresis detection spectrum of a sample 4 randomly extracted from an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention;
FIG. 7 is an electrophoresis detection spectrum of a sample 5 randomly extracted from an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention;
FIG. 8 is an electrophoresis detection spectrum of a sample 6 randomly extracted from an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention;
FIG. 9 is an electrophoresis detection spectrum of a randomly extracted sample 7 in an F2 generation population generated by the hybridization F1 generation self-separation of an HR31 watermelon material sample and a PI189225 watermelon material sample in the method for identifying the resistance of watermelon anthracnose by using an SSR primer for identifying the resistance of watermelon anthracnose in the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Description of materials: the watermelon materials PI189225 and HR31, and their progeny are provided by agricultural academy of sciences in Wuhan City, wherein the two parent materials are an anti-anthracnose watermelon germplasm resource, and HR31 is a susceptible anthracnose watermelon germplasm resource.
EXAMPLE 1 development of SSR primers for identifying resistance to watermelon anthracnose
And (3) carrying out re-sequencing on the determined anti-anthracnose watermelon material PI189225 and the susceptible-anthracnose watermelon material HR31 by using a high-throughput sequencing technology, carrying out sequence comparison, and designing a PCR primer spanning the SSR locus by using DNAstar software. The forward primer sequence is clE-F: 5'-AGAGGAATGTGGTAATGAATAAACATCT-3' and the reverse primer sequence is clE-R: 5'-AATTGGCCCAAATATCCATATGAC-3'.
EXAMPLE 2 extraction of DNA from watermelon sample to be tested
Selecting 30-40 mg of tender tissue of each watermelon sample seed to be detected after 3-4 days of germination, placing the tender tissue in a 2.0ml pre-cooled centrifuge tube, quickly spreading about 0.5mg of DIECA crystal on the tissue, adding liquid nitrogen, crushing the sample into powder by using a tissue triturator, adding 700 mu L of 2% CTAB preheating buffer solution and 0.5mg of active carbon, carrying out water bath at 65 ℃ for 1h, adding 700 mu L of 24:1 chloroform-isoamylol, uniformly mixing, and centrifuging at 12,000rpm for 10 min; taking supernatant, adding into a 1.5mL centrifuge tube filled with 700 μ L isopropanol, mixing, placing in a refrigerator at-20 deg.C for 30min, and centrifuging at 12,000rpm for 10 min; discarding supernatant, washing twice with 70% ethanol solution, drying under natural condition, adding 100 μ L ddH2And O, detecting the concentration after the solution is fully dissolved. The mixture is stored at-20 ℃ for use.
In this example, the modified CTAB method is used to extract the DNA of the sample to be tested:
before the sample is smashed into powder by a tissue triturator, a DIECA crystal is scattered on the tissue, and the DIECA crystal ensures that the tissue is fully ground, so that the DNA of the watermelon leaf tissue is prevented from being degraded.
After a sample is smashed into powder by a tissue triturator, CTAB preheating buffer solution and active carbon are added, then water bath is carried out, and the introduction of the active carbon prevents DNA degradation and prevents DNA adsorption on protein tissues.
EXAMPLE 3 detection of DNA of watermelon sample to be tested
Detecting the genomic DNA of the watermelon leaves by using 1 percent agarose gel electrophoresis, wherein the main band is clear and complete, and the watermelon leaves are not degraded and contain RNA; the ultraviolet spectrophotometer detects that A260/A280 is 1.85, which shows that the extracted DNA has higher purity and can meet the test requirements.
Example 4PCR reaction System and amplification procedure
Adding dNTP 0.25mmol/L, forward and reverse primers 0.2. mu. mol/L, TaqDNA polymerase 1.0U, 10 XPCR buffer (containing Mg2+), genome DNA of each watermelon sample 60ng, and sterilized double distilled water 15. mu.L; mixing and amplifying in PCR analyzer.
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s for 34 cycles; finally, extending for 10min at 72 ℃; storing at 4 deg.C;
example 5 capillary electrophoresis fluorescent detection analysis of PCR amplification products
The amplified 6-FAM and HEX fluorescence labeled PCR amplification products are diluted by 30 times with ultrapure water, and the TAMRA and ROX fluorescence labeled PCR amplification products are diluted by 10 times. Equal volumes of the 4 diluted solutions were mixed, and 1. mu.L of the mixture was pipetted into a deep well plate dedicated to a DNA analyzer. 0.1. mu.L of LIZ500 molecular weight internal standard and 8.9. mu.L of deionized formamide were added to each well of the plate. The sample is denatured for 5min at 95 ℃ on a PCR instrument, taken out, immediately placed on crushed ice, and cooled for more than 10 min. After being instantaneously centrifuged for 10s, the mixture is placed on a DNA analyzer for capillary electrophoresis fluorescence detection analysis.
Reading the data of the size of allelic variation of each sample by using fragment analysis software of a DNA analyzer; the parents of the positive primer and the reverse primer and F2 generation population capillary electrophoresis data generated by the self-separation of the two hybridized F1 generations are prepared into an electrophoresis map, and the electrophoresis map is shown in a figure 1. The SSR marker special primer can effectively distinguish whether the watermelon material has anthracnose resistance or not according to the map.
Example 6 identification of resistance to watermelon anthracnose by field Artificial inoculation
The method adopts a spraying method for identifying the resistance of the watermelon anthracnose through field artificial inoculation, and comprises the following specific steps: the cultured anthrax germ spore suspension is evenly inoculated on HR31 watermelon material sample, PI189225 watermelon material sample and F2 generation colony generated by F1 generation self-copulation separation in the table 1 by a small hand-held sprayer, and then samples (aseptic seedlings) from No.1 to No. 7 are randomly extracted. And (3) keeping the moisture for 48 hours under the dark condition of 24-26 ℃ and the relative humidity of 95-100%, and then giving a normal photoperiod. After 7 days of inoculation, the disease condition of the watermelon anthracnose is judged, and the disease classification and the reaction type of the disease are according to the regulations in agricultural industry Standard of the people's republic of China. The statistical results are as follows:
table 1: (List describes the results)
As can be seen from the comparison between Table 1 and FIGS. 1 to 9, the results obtained by the method for identifying resistance to watermelon anthracnose in examples 1 to 5 of the present invention are completely consistent with the results obtained by the field artificial inoculation resistance identification method.
Therefore, the SSR primers in the embodiment 1 of the invention can effectively distinguish whether the watermelon materials have anthracnose resistance or not.
The present invention is not limited to the above-described embodiments, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and such modifications and improvements are also considered to be within the scope of the present invention. Those not described in detail in this specification are within the skill of the art.
Sequence listing
<110> Wuhan City college of agricultural sciences
<120> SSR primer and method for identifying watermelon anthracnose resistance
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
agaggaatgt ggtaatgaat aaacatct 28
<210>2
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
aattggccca aatatccata tgac 24
Claims (9)
1. An SSR primer for identifying watermelon anthracnose resistance, which is characterized in that: the SSR primer has a forward primer of 5'-AGAGGAATGTGGTAATGAATAAACATCT-3' and a nucleotide sequence shown as SEQ ID NO.1 in a sequence table; the reverse primer is 5'-AATTGGCCCAAATATCCATATGAC-3', and the nucleotide sequence is shown as SEQ ID NO.2 in the sequence table.
2. A method of identifying watermelon anthracnose resistance using the SSR primer for identifying watermelon anthracnose resistance according to claim 1, wherein:
extracting DNA of a watermelon sample to be detected;
carrying out PCR amplification on the extracted DNA of the watermelon sample to be detected by using a fluorescence-labeled SSR primer;
and performing capillary electrophoresis fluorescence detection on the PCR amplification product, and judging whether the watermelon has anthracnose resistance according to the detection result.
3. The method of identifying watermelon anthracnose resistance according to claim 2, wherein: the method for extracting the DNA of the watermelon sample to be detected specifically comprises the following steps:
placing a watermelon sample to be detected in a centrifuge tube, adding liquid nitrogen, and smashing the sample to be detected by using a tissue triturator;
adding cetyl trimethyl ammonium bromide CTAB preheating buffer solution into a centrifuge tube, carrying out water bath, adding chloroform-isoamylol, uniformly mixing and centrifuging;
taking the supernatant, adding the supernatant into a centrifugal tube filled with isopropanol, uniformly mixing and centrifuging;
discarding the supernatant, washing the precipitate with 70% ethanol solution, drying, and adding double distilled water.
4. The method of identifying watermelon anthracnose resistance according to claim 3, wherein: before the sample to be tested is smashed by a tissue triturator, diethyl dithiocarbamate DIECA crystals are scattered on the sample to be tested.
5. The method of identifying watermelon anthracnose resistance according to claim 3, wherein: before the water bath, activated carbon was added to the centrifuge tube.
6. The method of identifying watermelon anthracnose resistance according to claim 2, wherein: the PCR amplification of the extracted DNA of the watermelon sample to be detected by using the SSR primer comprises the following steps:
adding deoxyribonucleoside triphosphate, a fluorescence-labeled forward primer and a reverse primer, TaqDNA polymerase and a PCR buffer solution containing Mg2+ into a PCR tube, uniformly mixing the DNA of a watermelon sample to be detected and double distilled water, and placing the mixture on a PCR instrument for PCR amplification.
7. The method of identifying watermelon anthracnose resistance according to claim 6, wherein: the reaction process of the PCR amplification comprises the following steps: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s for 34 cycles; extending for 10min at 72 ℃, and storing for later use at 4 ℃.
8. The method of identifying watermelon anthracnose resistance according to claim 2, wherein: the method comprises the following steps of performing capillary electrophoresis fluorescence detection on a PCR amplification product, and judging whether the watermelon has anthracnose resistance according to a capillary electrophoresis fluorescence detection result:
diluting PCR amplification products of 6-FAM fluorescent marker and HEX fluorescent marker by 30 times with ultrapure water respectively, and diluting PCR amplification products of TAMRA fluorescent marker and ROX fluorescent marker by 10 times with ultrapure water respectively;
respectively taking the diluted solutions of the 4PCR amplification products with the same volume, mixing to prepare a mixed solution, adding the mixed solution into a deep hole plate special for a DNA analyzer, and respectively adding an LIZ500 molecular weight internal standard and deionized formamide into each hole in the deep hole plate special for the DNA analyzer;
denaturing at 95 ℃ on a PCR analyzer, taking out and cooling, instantly centrifuging, and placing on a DNA analyzer for capillary electrophoresis detection;
using fragment analysis software of a DNA analyzer to analyze images, and making capillary electrophoresis data into an electrophoresis detection map; and judging whether the watermelon has the anthracnose resistance or not according to the electrophoresis detection map.
9. The method of identifying watermelon anthracnose resistance according to claim 2, wherein: the watermelon sample to be detected comprises a disease-resistant parent, a disease-susceptible parent and a filial generation of the disease-resistant parent and the disease-susceptible parent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810826669.4A CN110760602B (en) | 2018-07-25 | 2018-07-25 | SSR primer and method for identifying watermelon anthracnose resistance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810826669.4A CN110760602B (en) | 2018-07-25 | 2018-07-25 | SSR primer and method for identifying watermelon anthracnose resistance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110760602A true CN110760602A (en) | 2020-02-07 |
| CN110760602B CN110760602B (en) | 2022-12-27 |
Family
ID=69327243
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810826669.4A Active CN110760602B (en) | 2018-07-25 | 2018-07-25 | SSR primer and method for identifying watermelon anthracnose resistance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110760602B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814804A (en) * | 2005-11-25 | 2006-08-09 | 华东理工大学 | Watermelon anthrax bacteria detecting kit and its detecting method |
| CN102220315A (en) * | 2011-04-15 | 2011-10-19 | 北京市农林科学院 | Watermelon complete genomic sequence information based analyzed and developed SSR core primer combinations and application thereof |
| US20150101072A1 (en) * | 2013-10-04 | 2015-04-09 | Vilmorin & Cie | Combining fusarium 2 resistance gene (fon2) and red flesh in watermelon |
| CN107164545A (en) * | 2017-07-19 | 2017-09-15 | 北京市农林科学院 | The specificity identification method of variety of watermelon " capital is beautiful " |
-
2018
- 2018-07-25 CN CN201810826669.4A patent/CN110760602B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814804A (en) * | 2005-11-25 | 2006-08-09 | 华东理工大学 | Watermelon anthrax bacteria detecting kit and its detecting method |
| CN102220315A (en) * | 2011-04-15 | 2011-10-19 | 北京市农林科学院 | Watermelon complete genomic sequence information based analyzed and developed SSR core primer combinations and application thereof |
| US20150101072A1 (en) * | 2013-10-04 | 2015-04-09 | Vilmorin & Cie | Combining fusarium 2 resistance gene (fon2) and red flesh in watermelon |
| CN107164545A (en) * | 2017-07-19 | 2017-09-15 | 北京市农林科学院 | The specificity identification method of variety of watermelon " capital is beautiful " |
Non-Patent Citations (2)
| Title |
|---|
| C. MUJAJU 等: "Genetic diversity in watermelon (Citrullus lanatus) landraces", 《HEREDITAS》 * |
| 周鹏 等: "籽用西瓜SSR体系的建立与优化", 《内蒙古农业大学学报( 自然科学版)》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110760602B (en) | 2022-12-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| St. Laurent et al. | Rootstock genotype succession influences apple replant disease and root-zone microbial community composition in an orchard soil | |
| Moreno et al. | The use of RAPD markers for identification of cultivated grapevine (Vitis vinifera L.) | |
| Gayoso et al. | Assessment of real-time PCR as a method for determining the presence of Verticillium dahliae in different Solanaceae cultivars | |
| Sasaki et al. | Effects of plant genotype and nitrogen level on bacterial communities in rice shoots and roots | |
| Ondieki et al. | Morphological and genetic diversity of Rhizobia nodulating cowpea (Vigna unguiculata L.) from agricultural soils of lower eastern Kenya | |
| CN109486970B (en) | Loop-mediated isothermal amplification detection primer group, detection method and detection kit for xanthomonas oryzae | |
| Abid et al. | Genetic relationship and diversity analysis of faba bean (Vicia faba L. var. Minor) genetic resources using morphological and microsatellite molecular markers | |
| Zhang et al. | A novel strategy for identification of 47 pomegranate (Punica granatum) cultivars using RAPD markers | |
| Sa et al. | Identification of genetic variations of cultivated and weedy types of Perilla species in Korea and Japan using morphological and SSR markers | |
| Aban et al. | Molecular, morphological and pathogenic diversity of Sclerotinia sclerotiorum isolates from common bean (Phaseolus vulgaris) fields in Argentina | |
| Zhou et al. | Development of a loop‐mediated isothermal amplification method for the rapid detection of Venturia carpophila on peach | |
| Doumbia et al. | Assessment of cowpea germplasms from Ghana and Mali using simple sequence repeat (SSR) markers | |
| Oteng-Frimpong et al. | Assessing the genetic diversity of 48 groundnut (Arachis hypogaea L.) genotypes in the Guinea savanna agro-ecology of Ghana, using microsatellite-based markers | |
| Lin et al. | Genetic diversity and identification of race 3 of Fusarium oxysporum f. sp. lactucae in Taiwan | |
| CN108660247B (en) | Primer SmemboI-2 for identifying purity of solanum torvum third eggplant based on SNP marker and application | |
| KR101605530B1 (en) | A method for identifying strawberry varieties using microsatellites markers | |
| CN111349717B (en) | SSR (simple sequence repeat) markers of sweet cherry stock resources and fingerprint spectrum database thereof | |
| CN108707692B (en) | Method and primer for rapidly identifying purity of hybrid seed of watermelon variety' Hubei watermelon No. 16 | |
| KR101174409B1 (en) | A Method for Identifying Cucumber Varieties using Microsatellites Markers | |
| CN110760602B (en) | SSR primer and method for identifying watermelon anthracnose resistance | |
| Li et al. | Pathogenicity and genetic diversity of Fusarium oxysporum causing soybean root rot in northeast China | |
| CN108004346A (en) | Wheat cdna Yr10 molecular labelings and its application in Stripe rust resistance wheat is screened | |
| Singh et al. | Genetic purity assessment in linseed (Linum usitatissimum L.) varieties using microsatellite markers | |
| Das et al. | Assessment of community level physiological profiles and molecular diversity of soil bacteria under different cropping systems | |
| Ning et al. | Genetic diversity analysis of sedges (Carex spp.) in Shandong, China based on inter-simple sequence repeat |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |