CN113604596A - KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof - Google Patents
KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof Download PDFInfo
- Publication number
- CN113604596A CN113604596A CN202110915013.1A CN202110915013A CN113604596A CN 113604596 A CN113604596 A CN 113604596A CN 202110915013 A CN202110915013 A CN 202110915013A CN 113604596 A CN113604596 A CN 113604596A
- Authority
- CN
- China
- Prior art keywords
- zym
- cucumber
- primer
- sequence
- kasp
- 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.)
- Pending
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
Abstract
The invention relates to the technical field of molecular biology and cucumber breeding, in particular to a KASP primer for detecting cucumber courgette yellows mosaic virus disease resistance gene zym and application thereof. The invention provides KASP primers and a kit containing the KASP primers, which can be used for detecting zym gene with different genotypes of 86bp and 99 bp. The KASP primer or kit can be used for identifying whether cucumber contains ZYMV disease-resistant gene Zym, and distinguishing cucumber containing Zym gene and resistant to ZYMV from cucumber containing mutant Zym gene and susceptible to ZYMV. Compared with the prior art, the invention can rapidly, accurately and high-flux perform ZYMV resistance identification in the seedling stage of the cucumber, greatly reduces the workload of manual inoculation and field resistance identification in the seedling stage, can improve the breeding efficiency of the cucumber, quickens the breeding process and saves the cost. The KASP primer and the kit can be applied to the identification of materials such as cucumber germplasm resources, various parents, hybrid seeds and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology and crop breeding, in particular to a KASP primer for detecting cucumber courgette yellows mosaic virus disease resistance gene zym and application thereof.
Background
Cucumber (Cucumis sativus L.) belongs to annual climbing herbaceous plants of Cucurbitaceae, and is originally produced in India, is the third vegetable crop internationally, and is also one of the main cultivated vegetables in China. Cucumber small Zucchini Yellow Mosaic Virus (ZYMV) is one of the main viruses harming cucumbers, is widely spread in sunlight greenhouses in recent years, seriously influences the yield and quality of cucumbers, and can effectively resist ZYMV and reduce loss when used for cultivating antiviral varieties.
ZYMV is a potyvirus, and can be transmitted by aphid to infect Cucurbitaceae, Amaranthaceae, Chenopodiaceae, Leguminosae, etc. When cucumber is infected with ZYMV, the plants are yellowed and have mosaic shape and bubble or tumor-shaped protrusion, and when the disease is serious, the plants are dwarfed, the leaves are shriveled, the fruits are malformed, and even the plants die. However, no effective chemical agent or other method is available in actual production to prevent and treat ZYMV. The cultivation of new antiviral cucumber varieties is a fundamental approach for preventing and treating ZYMV.
The conventional breeding is time-consuming and labor-consuming, has higher environmental requirement and higher difficulty and has certain limitation. With the development of modern molecular biotechnology in recent years, a new thought is provided for plant breeding, and the breeding process is accelerated. The molecular marker assisted selection is a selection of target traits on a DNA level, has the advantages of no environmental influence, no interference of recessive relation of alleles, accurate and reliable selection result and the like, and is more and more favored by breeders. The common molecular markers are RFLP, RAPD, AFLP, SSR, CAPS and the like, and are widely applied to cucumber breeding, but the markers are complex to operate, high in experimental requirement, low in detection efficiency, not suitable for large-sample and high-flux detection. In order to meet the breeding requirement, it is important to establish a high-efficiency, convenient and accurate detection method.
The SNP marker is taken as a 3 rd generation molecular marker, is currently accepted as a molecular marker technology with a great application prospect, and a competitive Allele Specific PCR (KASP) technology developed based on the SNP marker has the advantages of high throughput, time saving, convenience and the like. The method is mainly characterized in that 3 specific primers are adopted as primers, wherein 2 upstream primers are adopted, 3 'ends are allelic variant bases, 5' ends are added with universal fluorescent adaptor sequences, universal downstream primers are common primers, conventional PCR amplification is carried out, and fluorescent signal detection is carried out by an end point method.
Many studies have been made on ZYMV by scholars at home and abroad, and in 1973, Lisa et al isolated from Cucurbita pepo and described ZYMV for the first time. The genetic regularity of ZYMV was studied by Provvidenti et al, and it was thought that the ZYMV resistance gene was controlled by a pair of recessive genes zym/zym. Amano et al resistance gene zym of ZYMVA192-18Located in the 50kb interval of cucumber chromosome VI, and through gene sequence analysis between disease-resistant and susceptible parents, it shows that the Vacuolar protein organizing-associated protein 4-like (VPS4-like) gene (CsA6G152960.1) is most likely to be a candidate ZYMV disease-resistant gene which is possibly related to virus replication and movement among cells. The disease-resistant and disease-susceptible parents have two SNP sites at the first exon of the gene, which are respectively positioned at the 86bp (T/C) and the 99bp (G/A). The disease-resistant parent is C at the 86bp and A at the 99 bp; the susceptible parent is T at 86bp and G at 99 bp. The two SNP loci make it possible to detect the gene by using KASP technology, greatly improve the breeding efficiency of disease-resistant varieties and shorten the breeding period.
Disclosure of Invention
The invention aims to provide a KASP primer for detecting cucumber courgette yellows mosaic virus disease resistance gene Zym and application thereof, which can be used for identifying the cucumber courgette yellows mosaic virus disease resistance gene Zym and whether the cucumber contains Zym gene, distinguishing the cucumber containing Zym gene ZYMV resistance and the cucumber containing mutant gene Zym sensing ZYMV, distinguishing the cucumber containing Zym gene homozygous ZYMV resistance (Zym/Zym), the cucumber containing mutant gene Zym homozygous ZYMV sensing (Zym/Zym) and the cucumber containing both Zym and Zym gene heterozygous ZYMV sensing (Zym/Zym), and further breeding the cucumber variety containing Zym gene ZYMV resistance.
The overall technical concept of the invention is as follows:
a KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym, wherein the KASP primer sequence comprises:
forward primer zym-86-FT: 5'-GCAATTACGCAAAAGCCTT-3', the sequence is shown as SEQ No. 1;
forward primer zym-86-FC: 5'-GCAATTACGCAAAAGCCTC-3', the sequence of which is shown in SEQ No. 2;
reverse primer zym-99-RC: 5'-TTGAAGTACTCCAAGGCGTTC-3', the sequence of which is shown in SEQ No. 3;
reverse primer zym-99-RT: 5'-TTGAAGTACTCCAAGGCGTTT-3', the sequence of which is shown in SEQ No. 4;
the forward primers zym-86-FT and zym-86-FC are respectively connected with different tag sequences;
the reverse primer zym-99-RC and the reverse primer zym-99-RT are respectively connected with different tag sequences.
When the forward primer zym-86-FT and the reverse primer zym-99-RC are synthesized, the sequences are added at the 5' ends of the two primers: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 5; when the forward primer zym-86-FC and the reverse primer zym-99-RT are synthesized, the sequences are added at the 5' ends of the two primers: 5'-GAAGGTCGGAGTCAACGGATT-3', and the sequence is shown in SEQ No. 6.
The KASP primer sequence is as follows:
forward primer 86-FT: 5'-GAAGGTGACCAAGTTCATGCTGCAATTACGCAAAAGCCTT-3', the sequence of which is shown in SEQ No. 7;
forward primer 86-FC: 5'-GAAGGTCGGAGTCAACGGATTGCAATTACGCAAAAGCCTC-3', the sequence of which is shown in SEQ No. 8;
reverse primer 99-RC: 5'-GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3', the sequence of which is shown in SEQ No. 9;
reverse primer 99-RT: 5'-GAAGGTCGGAGTCAACGGATTTTGAAGTACTCCAAGGCGTTT-3', the sequence is shown in SEQ No. 10.
The KASP primer also comprises:
reverse primer 86-R: 5'-CTTGGGATTTTTCTCATACTTCA-3', the sequence is shown as SEQ No. 11;
forward primer 99-F: 5'-CTATTGAATACGTGAAGCAGGC-3', the sequence is shown in SEQ No. 12.
The kit for detecting the cucumber courgette yellowing mosaic virus disease resistance gene zym comprises the KASP primers, wherein the forward primer 86-FT, the forward primer 86-FC and the reverse primer 86-R are a pair of KASP primers; the reverse primer 99-RC, the reverse primer 99-RT and the forward primer 99-F are a pair of KASP primers.
The kit for detecting the cucumber courgette yellows mosaic virus disease resistance gene zym further comprises:
tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 5;
a tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3', the sequence of which is shown in SEQ No. 6;
the two tag sequences: the kit comprises a tag sequence A and a tag sequence B, wherein the tag sequence A and the tag sequence B are respectively connected with different fluorophores;
the complementary sequences of the two tag sequences are connected with BHQ quenching genes.
The application of the KASP primer for detecting the cucumber small zucchini yellow mosaic virus disease resistance gene zym or the kit for detecting the cucumber small zucchini yellow mosaic virus disease resistance gene zym in at least one of the following a-e:
a. identifying zym disease-resistant gene of cucumber courgette yellows mosaic virus;
b. identifying whether the cucumber contains the cucumber courgette yellows mosaic virus disease resistance gene zym;
c. identifying or differentiating between ZYMV-resistant cucumbers containing Zym gene and ZYMV-sensitive cucumbers containing mutated Zym gene;
d. differentiating cucumber homozygous for ZYMV resistant containing Zym gene (Zym/Zym), cucumber homozygous for ZYMV sensitive containing mutant gene Zym (Zym/Zym), and cucumber heterozygous for ZYMV sensitive containing both Zym gene and Zym gene (Zym/Zym);
e. and (5) breeding the cucumber with ZYMV resistance.
The application of zym in identifying whether cucumber courgette yellows mosaic virus disease resistance gene is contained in cucumber is to amplify a cucumber sample to be detected by using KASP primer for detecting the cucumber courgette yellows mosaic virus disease resistance gene zym or kit for detecting the cucumber courgette yellows mosaic virus disease resistance gene zym and detect and analyze the amplified product.
The application of differentiating ZYMV-resistant cucumber containing Zym gene and ZYMV-sensitive cucumber containing mutant Zym gene is to amplify cucumber sample to be detected by using KASP primer for detecting cucumber courgette yellows mosaic virus disease resistance gene Zym or kit for detecting cucumber courgette yellows mosaic virus disease resistance gene Zym, and detect and analyze the amplified product.
The application of differentiating homozygous ZYMV-resistant cucumber (Zym/Zym) containing Zym gene, homozygous ZYMV-resistant cucumber (Zym/Zym) containing mutant gene Zym and heterozygous ZYMV-resistant cucumber (Zym/Zym) containing both Zym gene and Zym gene is to amplify cucumber samples to be detected by using KASP primers for detecting cucumber courgette yellowed mosaic virus disease resistance gene Zym or a kit for detecting cucumber courgette yellowed mosaic virus disease resistance gene Zym, and detect and analyze amplified products.
The invention achieves the substantive characteristics and obvious technical progress that:
by using the KASP primer and the kit provided by the invention, when cucumber plants are in the seedling stage, the detection of the cucumber courgette yellowing mosaic virus disease resistance gene zym can be rapidly, accurately and high-flux completed by using a small amount of leaf tissues, so that the workload of manual inoculation and field disease resistance identification in the seedling stage is greatly reduced. The KASP primer and the kit can be applied to the identification of materials such as cucumber planting resources, various parents, hybrids and the like, and the application of the KASP primer and the kit can improve the ZYMV-resistant breeding efficiency of cucumbers, accelerate the breeding process and save the cost.
Drawings
FIG. 1 shows the result of detecting the 86bp SNP site KASP marker of zym gene.
In the figure, the abscissa and ordinate values both represent fluorescence signal values, wherein the ordinate represents FAM fluorescence signal values and the abscissa represents HEX fluorescence signal values; amplification signals for homozygous anti-ZYMV material TMG1, represented by black triangles near the horizontal axis in the figure; the amplification signals of the heterozygous ZYMV-sensitive material TMG1-9930-F1 are represented by black diamonds in the middle of the graph; the amplification signal of ZYMV material 9930 which is homozygous and is represented by an inverted triangle near the vertical axis in the figure; the black dots near the origin represent the amplification signal of the negative control (no DNA sample added).
FIG. 2 shows the result of detecting the KASP marker at the 99bp SNP site of zym gene.
In the figure, the abscissa and ordinate values both represent fluorescence signal values, wherein the ordinate represents FAM fluorescence signal values and the abscissa represents HEX fluorescence signal values; amplification signals for homozygous anti-ZYMV material TMG1, represented by black triangles near the horizontal axis in the figure; the amplification signals of the heterozygous ZYMV-sensitive material TMG1-9930-F1 are represented by black diamonds in the middle of the graph; the amplification signal of ZYMV material 9930 which is homozygous and is represented by an inverted triangle near the vertical axis in the figure; the black dots near the origin represent the amplification signal of the negative control (no DNA sample added).
FIG. 3 shows the results of typing ZYMV-resistant cucumbers and ZYMV-susceptible cucumbers by using KASP primer for detecting the 8bp SNP site of zym gene.
In the figure, the abscissa and ordinate values both represent fluorescence signal values, wherein the ordinate represents FAM fluorescence signal values and the abscissa represents HEX fluorescence signal values; the amplification signals of the homozygous ZYMV-resistant cucumber material are represented by black triangles near the horizontal axis; amplification signals of heterozygous ZYMV-sensitive cucumber material, indicated by the black diamonds in the middle of the figure; the amplification signals of cucumber material homozygous for ZYMV are indicated by the inverted triangle near the vertical axis in the figure; the black dots near the origin represent the amplification signal of the negative control (no DNA sample added).
FIG. 4 shows the results of typing ZYMV-resistant cucumbers and ZYMV-susceptible cucumbers by using KASP primer for detecting the 99bp SNP site of zym gene.
In the figure, the abscissa and ordinate values both represent fluorescence signal values, wherein the ordinate represents FAM fluorescence signal values and the abscissa represents HEX fluorescence signal values; the amplification signals of the homozygous ZYMV-resistant cucumber material are represented by black triangles near the horizontal axis; amplification signals of heterozygous ZYMV-sensitive cucumber material, indicated by the black diamonds in the middle of the figure; the amplification signals of cucumber material homozygous for ZYMV are indicated by the inverted triangle near the vertical axis in the figure; the black dots near the origin represent the amplification signal of the negative control (no DNA sample added).
Detailed Description
The present invention is further described with reference to the following examples, which are not intended to limit the scope of the present invention, and the claims are not to be interpreted as limiting the scope of the present invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The reagents (Wuhan city peptide Biotech Co., Ltd.) used in the examples described below and the like are commercially available unless otherwise specified.
Cucumber material used in the following examples: TMG1 (homozygous ZYMV-resistant material containing Zym gene), 9930 (homozygous ZYMV-resistant material containing Zym gene), and TMG1-9930-F1 (heterozygous ZYMV-resistant material containing both Zym gene and Zym gene), the public can ask the institute of economic crops of the academy of agriculture and forestry, Hebei to repeat the experiment.
The KASP genotyping method is simple to operate, and only needs to add the specific KASP Primer mix and the general KASP Master mix into a PCR reaction hole containing a DNA sample for PCR amplification, and a final result is analyzed by a fluorescence detector for PCR products.
The invention provides KASP primers capable of detecting two SNP sites of zym gene based on KASP technology, and can identify zym gene on high-throughput molecular marker detection platform by combining Touchdown PCR method with strict conditions.
Specifically, the applicant, based on previous studies, controlled resistance of cucumber to ZYMV by a single recessive gene, had two SNP sites at the first exon of the zym candidate gene VPS4-like for the anti-ZYMV parent and the susceptible ZYMV parent, respectively at the 86 th base (T/C) and the 99 th base (G/A). The bases at position 86 of the anti-ZYMV parent and the susceptible ZYMV parent are C and T, respectively, and the bases at position 99 are A and G, respectively. A large number of experiments are carried out aiming at the two SNP sites, and two specific forward primers and a universal reverse primer for detecting the 86 th (T/C) SNP site of the gene, and a universal forward primer and two specific reverse primers for detecting the 99 th (G/A) SNP site are screened and determined.
In particular embodiments, the KASP primer designed for the SNP site needs to include two specific forward (or reverse) primers and one universal reverse (or forward) primer.
For example, the KASP primers for detecting the 86 th (T/C) SNP site of zym gene provided by the application comprise forward primers zym-86-FT: 5'-GCAATTACGCAAAAGCCTT-3' and forward primer zym-86-FC: 5'-GCAATTACGCAAAAGCCTC-3', the last base of the two primers is different only at the 3 'end, i.e. the corresponding SNP site, and different tag sequences are respectively connected to the 5' ends of the two forward primers, such as tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', and tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3' are provided.
For example, the KASP primers for detecting the 99 th (G/A) SNP site of zym gene provided by the present application include reverse primer zym-99-RC: 5'-TTGAAGTACTCCAAGGCGTTC-3' and reverse primer zym-99-RT: 5'-TTGAAGTACTCCAAGGCGTTT-3', the last base of the two primers only differs from the last base of the 3 'end, i.e. the corresponding SNP site, and the 5' ends of the two reverse primers are respectively linked with different tag sequences, such as tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', and tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3' are provided.
For example, the forward primer 86-FT provided herein: 5' -GAAGGTGACCAAGTTCATGCTGCAATTACGCAAAAGCCTT-3', and forward directionSubstance 86-FC:
(ii) a Reverse primer 99-RC: 5' -GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3', and reverse primer 99-RT: 5' -for example, the KASP primers for detecting the 86 th (T/C) SNP site of zym gene provided herein include forward primers zym-86-FT: 5'-GCAATTACGCAAAAGCCTT-3' and forward primer zym-86-FC: 5'-GCAATTACGCAAAAGCCTC-3', the last base of the two primers is different only at the 3 'end, i.e. the corresponding SNP site, and different tag sequences are respectively connected to the 5' ends of the two forward primers, such as tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', and tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3' are provided.
. Wherein the underlined sequence is the added tag sequence A and the wavy line is the tag sequence B.
In a specific embodiment, the length of the PCR product amplified by the KASP primer is preferably between 80bp and 150bp, and the sequence can be changed under the condition of meeting the requirement. For example, the reverse primer 86-R for detecting the 86 th (T/C) SNP site of zym gene provided by the application: 5'-CTTGGGATTTTTCTCATACTTCA-3', the amplification length is 83 bp; a forward primer 99-F for detecting the 99 th (G/A) SNP site of zym gene: 5'-CTATTGAATACGTGAAGCAGGC-3', the amplified length is 94 bp.
Further, in a specific embodiment, the kit provided by the present application further comprises a fluorescent probe a, a fluorescent probe B, a quenching probe a and a quenching probe B, wherein the sequence of the fluorescent probe a is identical to that of the tag sequence a, is 5'-GAAGGTGACCAAGTTCATGCT-3', and 1 fluorophore FAM is linked to the 5 ' end of the fluorescent probe a; the sequence of the fluorescent probe B is identical to that of the tag sequence B, is 5'-GAAGGTCGGAGTCAACGGATT-3', and is connected with 1 fluorescent group HEX at the 5 ' end. The sequence of the quenching probe A is complementary to that of the tag sequence A, is 5'-CTTCCACTGGTTCAAGTACGA-3', and has a sequence shown as SEQ No. 13; the sequence of the quenching probe B is complementary to the label sequence B, is 5'-CTTCCAGCCTCAGTTGCCTAA-3', and has a sequence shown in SEQ No. 14; meanwhile, quenching gene BHA is connected to the 3' ends of both quenching probe A and quenching probe B.
For example, the above-mentioned fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B can be obtained from PARMS (PRO2.0) kit from peptide Biotech, Inc. in the market of Wuhan.
The KASP primer for detecting cucumber courgette yellowed mosaic virus disease resistance gene Zym or the kit for detecting cucumber courgette yellowed mosaic virus disease resistance gene Zym provided by the application can be used for identifying whether the cucumber contains Zym gene, and can distinguish the cucumber containing Zym gene homozygous ZYMV resistant (Zym/Zym), the cucumber containing mutant gene Zym homozygous ZYMV (Zym/Zym) and the cucumber containing both Zym gene and Zym gene heterozygous ZYMV (Zym/Zym).
In a specific embodiment, the present application provides a method for identifying whether the zym gene is contained in cucumber, comprising amplifying the DNA of a cucumber sample to be identified by using the KASP primer provided by the present application, detecting and analyzing the amplified product.
In particular embodiments, the KASP provided herein: for example, the forward primer 86-FT: 5' -GAAGGTGACCAA GTTCATGCTGCAATTACGCAAAAGCCTT-3', and forward primer 86-FC:
and a reverse primer 86-R: 5'-CTTGGGATTTTTCTCATACTTCA-3', respectively; or the reverse primer 99-RC: 5' -GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3', and reverse primer 99-RT:
and forward primer 99-F: 5'-CTATTGAATACGTGAAGCAGGC-3' are provided. After PCR amplification, if only HEX fluorescent signals are detected in the reaction, determining that the cucumber sample to be detected contains zym genes; if only FAM fluorescent signals are detected, determining that the cucumber sample contains mutated Zym genes; if the HEX fluorescence signal and the FAM fluorescence signal are detected simultaneously, the cucumber sample is determined to contain the Zym gene and the Zym gene simultaneously.
In a specific embodiment, the cucumber sample to be detected may be DNA of any one of leaves, roots, stems, flowers, fruits and seeds of a cucumber plant.
In another embodiment, the present application provides a method for differentiating between cucumber resistant to ZYMV and cucumber sensitive to ZYMV, and the use of differentiating between cucumber homozygous resistant to ZYMV containing Zym gene (Zym/Zym), cucumber homozygous sensitive to ZYMV containing mutant gene Zym (Zym/Zym), and cucumber heterozygous sensitive to ZYMV containing both Zym gene and Zym gene (Zym/Zym), comprising amplifying DNA of a cucumber sample to be identified using the KASP primers provided herein, and detecting and analyzing the amplified product.
In particular embodiments, the KASP provided herein: for example, the forward primer 86-FT: 5' -GAAGGTGACCAA GTTCATGCTGCAATTACGCAAAAGCCTT-3', and forward primer 86-FC:
and a reverse primer 86-R: 5'-CTTGGGATTTTTCTCATACTTCA-3', respectively; or the reverse primer 99-RC: 5' -GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3', and reverse primer 99-RT:
and forward primer 99-F: 5'-CTATTGAATACGTGAAGCAGGC-3' are provided. PCR amplificationThen, if only HEX fluorescent signals are detected in the reaction, determining the cucumber sample to be detected as ZYMV-resistant cucumber; and if only FAM signals are detected or HEX fluorescent signals and FAM fluorescent signals are detected at the same time, determining that the cucumber sample to be detected is cucumber with ZYMV feeling.
In particular embodiments, the KASP provided herein: for example, the forward primer 86-FT: 5' -GAAGGTGACCAA GTTCATGCTGCAATTACGCAAAAGCCTT-3', and forward primer 86-FC:
and a reverse primer 86-R: 5'-CTTGGGATTTTTCTCATACTTCA-3', respectively; or the reverse primer 99-RC: 5'-GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3', and reverse primer 99-RT:
and forward primer 99-F: 5'-CTATTGAATACGTGAAGCAGGC-3' are provided. If only HEX fluorescent signals are detected through amplification, the cucumber sample to be detected is determined to be a homozygous ZYMV-resistant sample containing zym genes; if HEX and FAM signals are detected simultaneously, the cucumber sample is determined to be cucumber with heterozygous ZYMV containing both Zym gene and Zym gene; if only FAM fluorescent signals are detected, the cucumber sample to be detected is determined to be cucumber containing mutant gene Zym homozygous ZYMV.
In a specific embodiment, the cucumber sample to be detected may be DNA of any one of leaves, roots, stems, flowers, fruits and seeds of a cucumber plant.
The following examples are for the purpose of illustration only and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are carried out according to conventional experimental conditions, e.g.Sambrook et al, handbook of molecular cloning experiments (Sambrook J & Russell DW, molecular: a laboratory Manual, 2001), or according to the conditions suggested by the manufacturer's instructions.
Example 1
Development of KASP markers
The DNA sequences of zym genes of the ZYMV resistant parent and the ZYMV sensitive parent are compared and analyzed, and two SNP sites are found at the first exon of the gene and are positioned at 86bp (T/C) and 99bp (G/A). The bases at position 86 of the anti-ZYMV parent and the susceptible ZYMV parent are C and T, respectively, and the bases at position 99 are A and G, respectively.
Designing KASP primer according to the SNP locus, wherein the primer sequence is as follows:
T/C at position 86:
forward primer 86-FT: 5' -GAAGGTGACCAAGTTCATGCTGCAATTACGCAAAAGCCTT-3’；
Forward primer 86-FC:
；
wherein the sequence shown by underlining is an added tag sequence A, and the sequence shown by wavy lines is a tag sequence B;
reverse primer 86-R: 5'-CTTGGGATTTTTCTCATACTTCA-3' are provided.
The primer test results are shown in FIG. 1.
G/A at position 99:
reverse primer 99-RC: 5' -GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3’；
Reverse primer 99-RT:
；
wherein the sequence shown by underlining is an added tag sequence A, and the sequence shown by wavy lines is a tag sequence B;
forward primer 99-F: 5'-CTATTGAATACGTGAAGCAGGC-3' are provided.
The primer test results are shown in FIG. 2.
Example 2
Amplification of molecular markers
The materials used in this example were ZYMV resistant TMG1, ZYMV sensitive material 9930, and TMG1-9930-F1 hybrid susceptible material prepared by crossing the two as parents, which are recognized in the cucumber breeding community. Collecting plant leaf tissue for genome DNA extraction.
The genomic DNA in the leaf of the above-mentioned material was extracted as required by the Plant DNA Isolation Kit (Kyowa Biotechnology Co., Ltd.), and the extracted DNA solution was diluted to a concentration of 50 to 100 ng/. mu.l and stored at-20 ℃.
According to the KASP-PARMS kit (Wuhan city scenery peptide Biotechnology limited) requirements, a PCR reaction system is configured, the total reaction volume is 10 mul, and the method comprises the following steps: 2X PARMS PCR Mix: 5 mu l of the solution; DNA extract (50-100 ng/. mu.l): 1 mul; specific primer 1: 0.15. mu.l (10 pmol/. mu.l); specific primers 2: 0.15. mu.l (10 pmol/. mu.l); the general primer is as follows: 0.4. mu.l (10 pmol/. mu.l); and ddH2O: 3.3. mu.l. Three technical repetitions are set.
The PCR reaction program is: 94 ℃ below zero: 15 minutes; 94 ℃ below zero: 20 seconds, 65 ℃ (0.8 ℃ drop per cycle): 1 minute, 10 cycles; 94 ℃ below zero: 20 seconds, 57 ℃: 1 minute, 28 cycles. PCR reactions were performed using an Applied Biosystems 7500Real-Time PCR System.
Example 3
Detection and analysis of amplification products
The PCR products were genotyped and data analyzed using the software available from the Applied Biosystems 7500Real-Time PCR System, where the ordinate values were set to represent FAM fluorescence signal values and the abscissa values to represent HEX fluorescence signal values.
The typing results of KASP primers designed based on the 86bp SNP site are shown in FIG. 1. In the graph, the amplification signal of the homozygous anti-ZYMV material TMG1 is represented by black triangles near the horizontal axis, and only the HEX fluorescence signal is detected; the black diamonds in the middle of the graph represent the amplification signals of the ZYMV material TMG1-9930-F1 with heterozygosity, and HEX and FAM fluorescence signals can be detected; the amplification signal of the ZYMV material 9930 of homozygous feeling is represented by an inverted triangle near the vertical axis in the figure, and only the FAM fluorescence signal is detected; the black circles near the origin indicate the amplification signal of the negative control (no DNA sample added), and neither HEX nor FAM fluorescence was evident. The fluorescent amplification signals of the homozygous ZYMV-resistant genotype and the homozygous ZYMV-sensitive genotype are respectively connected with the origin, and the closer the formed included angle is to the right angle, the better the typing effect is.
The results show that the KASP primer pair amplified signals for the cucumber material TMG1 were as expected near the horizontal axis, for 9930 were as expected near the vertical axis, and for TMG1-9930-F1 were as expected in the middle position.
The three genotypes can be obviously distinguished and clustered, and the KASP primer can be used and successfully designed.
The typing results of KASP primers designed based on the 99bp SNP site are shown in FIG. 2. In the graph, the amplification signal of the homozygous anti-ZYMV material TMG1 is represented by black triangles near the horizontal axis, and only the HEX fluorescence signal is detected; the black diamonds in the middle of the graph represent the amplification signals of the ZYMV material TMG1-9930-F1 with heterozygosity, and HEX and FAM fluorescence signals can be detected; the amplification signal of the ZYMV material 9930 of homozygous feeling is represented by an inverted triangle near the vertical axis in the figure, and only the FAM fluorescence signal is detected; the black circles near the origin indicate the amplification signal of the negative control (no DNA sample added), and neither HEX nor FAM fluorescence was evident. The fluorescent amplification signals of the homozygous ZYMV-resistant genotype and the homozygous ZYMV-sensitive genotype are respectively connected with the origin, and the closer the formed included angle is to the right angle, the better the typing effect is.
The results show that the KASP primer pair amplified signals for the cucumber material TMG1 were as expected near the horizontal axis, for 9930 were as expected near the vertical axis, and for TMG1-9930-F1 were as expected in the middle position.
The three genotypes can be obviously distinguished and clustered, and the KASP primer can be used and successfully designed.
Example 4
The KASP primer or the kit for detecting the cucumber zucchini yellow mosaic virus disease resistance gene zym is used for identifying and distinguishing the cucumber resisting ZYMV and the cucumber feeling ZYMV
The materials used in this example were ZYMV resistant TMG1, ZYMV sensitive material 9930, and TMG1-9930-F1 hybrid susceptible material prepared by crossing the two as parents, which are recognized in the cucumber breeding community. 338 parts of cucumber germplasm resources are widely collected (the specific typing results are shown in table 1). Collecting the leaf tissue of the cucumber plant for extracting genome DNA.
338 parts of the cucumber material was extracted as a template for PCR amplification using the method described in example 2; performing PCR amplification by using the KASP primer or the kit for detecting the cucumber courgette yellows mosaic virus disease resistance gene zym; PCR reactions were performed using an Applied Biosystems 7500Real-Time PCR System; the PCR products were genotyped and data analyzed using the software available from the Applied Biosystems 7500Real-Time PCR System, where the ordinate values were set to represent FAM fluorescence signal values and the abscissa values to represent HEX fluorescence signal values.
The results of KASP primer typing for detecting the 86bp SNP site of Zym gene revealed that the number of black triangles having the same band pattern as that of FIG. 3 near the horizontal axis was 60 (only HEX fluorescence signal was detected, and the cucumber material containing Zym gene and homozygous ZYMV-resistant gene was identified as C: C), the diamond shape having the same band pattern as that of FIG. 3 at the middle position was 91 (HEX and FAM signals were detected, and the cucumber material containing both Zym gene and Zym gene and heterozygous ZYMV was identified as C: T), and the inverted triangle having the same band pattern as that of FIG. 3 near the vertical axis was identified as 187 (only FAM fluorescence signal was detected, and the cucumber material containing homozygous ZYMV-resistant gene and mutated gene Zym was identified as T: T), and that the black dots near the origin of the band pattern as that of FIG. 3 were identified as negative control (no DNA sample was added).
The results of KASP primer typing for detecting the 99bp SNP site of Zym gene revealed that the number of black triangles having the same band pattern as that of FIG. 4 near the horizontal axis was 60 (only HEX fluorescence signal was detected, and the cucumber material containing Zym gene and homozygous ZYMV-resistant gene was obtained, the genotype was A: A), the diamond shape having the same band pattern as that of FIG. 3 at the middle position was 91 (HEX and FAM signals were detected, and the cucumber material containing both Zym gene and Zym gene and heterozygous ZYMV was obtained, the genotype was A: G), and the inverted triangle having the same band pattern as that of FIG. 3 near the vertical axis was 187 (only FAM fluorescence signal was detected, and the cucumber material containing homozygous ZYMV-resistant gene and mutant gene G: G) and that the black dots near the vertical axis of the band pattern as that of FIG. 3 were used to detect the amplification signal of negative control (no DNA sample was added).
The results show that the KASP primer or the kit for detecting the cucumber courgette yellows mosaic virus disease resistance gene zym can be used for accurately, quickly and high-flux identifying the ZYMV resistant cucumber and the cucumber feeling ZYMV.
TABLE 1 identification of typing results for ZYMV-resistant and ZYMV-susceptible cucumbers
Sequence listing
<110> institute of economic crops of academy of agriculture, forestry and science of Hebei province
<120> KASP primer for detecting cucumber small zucchini etiolating mosaic virus disease resistance gene zym and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 10
<211> 19
<212> DNA
<213> An Artificial Sequence
<400> 10
gcaattacgc aaaagcctt 19
<210> 2
<211> 19
<212> DNA
<213> An Artificial Sequence
<400> 2
gcaattacgc aaaagcctc 19
<210> 3
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 3
ttgaagtact ccaaggcgtt c 21
<210> 4
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 4
ttgaagtact ccaaggcgtt t 21
<210> 5
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 5
gaaggtgacc aagttcatgc t 21
<210> 6
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 6
gaaggtcgga gtcaacggat t 21
<210> 7
<211> 40
<212> DNA
<213> An Artificial Sequence
<400> 7
gaaggtgacc aagttcatgc tgcaattacg caaaagcctt 40
<210> 8
<211> 40
<212> DNA
<213> An Artificial Sequence
<400> 8
gaaggtcgga gtcaacggat tgcaattacg caaaagcctc 40
<210> 9
<211> 42
<212> DNA
<213> An Artificial Sequence
<400> 9
gaaggtgacc aagttcatgc tttgaagtac tccaaggcgt tc 42
<210> 10
<211> 42
<212> DNA
<213> An Artificial Sequence
<400> 10
gaaggtcgga gtcaacggat tttgaagtac tccaaggcgt tt 42
<210> 11
<211> 23
<212> DNA
<213> An Artificial Sequence
<400> 11
cttgggattt ttctcatact tca 23
<210> 12
<211> 22
<212> DNA
<213> An Artificial Sequence
<400> 12
ctattgaata cgtgaagcag gc 22
<210> 13
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 13
cttccactgg ttcaagtacg a 21
<210> 14
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 14
cttccagcct cagttgccta a 21
Claims (12)
1. The KASP primer for detecting the cucumber small zucchini yellow mosaic virus disease resistance gene zym is characterized in that the KASP primer sequence comprises:
forward primer zym-86-FT: 5'-GCAATTACGCAAAAGCCTT-3', the sequence is shown as SEQ No. 1;
forward primer zym-86-FC: 5'-GCAATTACGCAAAAGCCTC-3', the sequence of which is shown in SEQ No. 2;
reverse primer zym-99-RC: 5'-TTGAAGTACTCCAAGGCGTTC-3', the sequence of which is shown in SEQ No. 3;
reverse primer zym-99-RT: 5'-TTGAAGTACTCCAAGGCGTTT-3', the sequence of which is shown in SEQ No. 4;
the forward primers zym-86-FT and zym-86-FC are respectively connected with different tag sequences;
the reverse primer zym-99-RC and the reverse primer zym-99-RT are respectively connected with different tag sequences.
2. The KASP primer for detecting cucumber courgette yellows mosaic virus disease resistance gene zym according to claim 1, wherein the sequence is added to the 5' ends of the forward primer zym-86-FT and the reverse primer zym-99-RC when synthesizing the primers: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 5; when the forward primer zym-86-FC and the reverse primer zym-99-RT are synthesized, the sequences are added at the 5' ends of the two primers: 5'-GAAGGTCGGAGTCAACGGATT-3', and the sequence is shown in SEQ No. 6.
3. The KASP primer for detecting the cucumber courgette yellows mosaic virus disease resistance gene zym according to claim 1, wherein the KASP primer sequence is as follows:
forward primer 86-FT: 5'-GAAGGTGACCAAGTTCATGCTGCAATTACGCAAAAGCCTT-3', the sequence of which is shown in SEQ No. 7;
forward primer 86-FC: 5'-GAAGGTCGGAGTCAACGGATTGCAATTACGCAAAAGCCTC-3', the sequence of which is shown in SEQ No. 8;
reverse primer 99-RC: 5'-GAAGGTGACCAAGTTCATGCTTTGAAGTACTCCAAGGCGTTC-3', the sequence of which is shown in SEQ No. 9;
reverse primer 99-RT: 5'-GAAGGTCGGAGTCAACGGATTTTGAAGTACTCCAAGGCGTTT-3', the sequence is shown in SEQ No. 10.
4. The KASP primer for detecting cucumber courgette yellowing mosaic virus disease resistance gene zym as claimed in any one of claims 1-3, wherein the KASP primer further comprises:
reverse primer 86-R: 5'-CTTGGGATTTTTCTCATACTTCA-3', the sequence is shown as SEQ No. 11;
forward primer 99-F: 5'-CTATTGAATACGTGAAGCAGGC-3', the sequence is shown in SEQ No. 12.
5. Kit for detecting cucumber courgette yellowing mosaic virus disease resistance gene zym, comprising the KASP primer of any one of claims 1-4, wherein forward primer 86-FT, forward primer 86-FC, and reverse primer 86-R are a pair of KASP primers; the reverse primer 99-RC, the reverse primer 99-RT and the forward primer 99-F are a pair of KASP primers.
6. The kit according to claim 5, further comprising:
tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 5;
a tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3', the sequence of which is shown in SEQ No. 6;
the two tag sequences: the kit comprises a tag sequence A and a tag sequence B, wherein the tag sequence A and the tag sequence B are respectively connected with different fluorophores;
the complementary sequences of the two tag sequences are connected with BHQ quenching genes.
7. Use of the KASP primer according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 6 for identifying the cucumber courgette yellowed mosaic virus disease resistance gene zym.
8. Use of the KASP primer according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 6 for identifying whether cucumber has the cucurbita pepo yellowing mosaic virus disease resistance gene zym.
9. Use of a KASP primer according to any one of claims 1 to 4 or a kit according to any one of claims 5 to 6 for identifying or differentiating between cucumbers resistant to courgette yellow mosaic virus disease containing the disease resistance gene Zym and cucumbers susceptible to courgette yellow mosaic virus disease containing the mutant gene Zym.
10. Use of the KASP primer according to any of claims 1 to 4 or the kit according to any of claims 5 to 6 for differentiating between cucumbers homozygous for zucchini yellowing mosaic virus containing Zym gene (Zym/Zym), cucumbers homozygous for zucchini yellowing mosaic virus containing a mutant gene Zym (Zym/Zym), and cucumbers heterozygous for zucchini yellowing mosaic virus containing both the Zym gene and Zym gene (Zym/Zym).
11. Use of a KASP primer according to any one of claims 1 to 4 or a kit according to any one of claims 5 to 6 for the zym gene in breeding cucumber against cucurbita pepo yellowing mosaic virus disease.
12. Use of KASP primers according to any of claims 1 to 4 or of the kit according to any of claims 5 to 6 for amplifying a cucumber sample to be detected, detecting and analyzing the amplified product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110915013.1A CN113604596A (en) | 2021-08-10 | 2021-08-10 | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110915013.1A CN113604596A (en) | 2021-08-10 | 2021-08-10 | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113604596A true CN113604596A (en) | 2021-11-05 |
Family
ID=78340122
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110915013.1A Pending CN113604596A (en) | 2021-08-10 | 2021-08-10 | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604596A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923357A (en) * | 2019-12-31 | 2020-03-27 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
| CN116543837A (en) * | 2023-07-04 | 2023-08-04 | 北京市农林科学院 | Genotype comparison method and device based on fluorescent signal platform |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112546A (en) * | 2015-09-23 | 2015-12-02 | 中国农业科学院作物科学研究所 | Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer |
| CN111961749A (en) * | 2020-09-14 | 2020-11-20 | 河北省农林科学院经济作物研究所 | KASP primer for detecting tomato yellow leaf curl virus disease resistance genes Ty-3 and Ty-3a and application thereof |
| CN111961750A (en) * | 2020-09-14 | 2020-11-20 | 河北省农林科学院经济作物研究所 | KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof |
-
2021
- 2021-08-10 CN CN202110915013.1A patent/CN113604596A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112546A (en) * | 2015-09-23 | 2015-12-02 | 中国农业科学院作物科学研究所 | Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer |
| CN111961749A (en) * | 2020-09-14 | 2020-11-20 | 河北省农林科学院经济作物研究所 | KASP primer for detecting tomato yellow leaf curl virus disease resistance genes Ty-3 and Ty-3a and application thereof |
| CN111961750A (en) * | 2020-09-14 | 2020-11-20 | 河北省农林科学院经济作物研究所 | KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| MASASHI AMANO ET AL.: "High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber", 《THEOR APPL GENET》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923357A (en) * | 2019-12-31 | 2020-03-27 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
| CN116543837A (en) * | 2023-07-04 | 2023-08-04 | 北京市农林科学院 | Genotype comparison method and device based on fluorescent signal platform |
| CN116543837B (en) * | 2023-07-04 | 2024-01-26 | 北京市农林科学院 | Genotype comparison method and device based on fluorescent signal platform |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2511381B1 (en) | Methods for sequence-directed molecular breeding | |
| CN111961750A (en) | KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof | |
| CN111961749B (en) | KASP primer for detecting tomato yellow leaf curl virus disease-resistant genes Ty-3 and Ty-3a and application thereof | |
| US20080028480A1 (en) | Markers for Salinity Tolerance in Wheat Plants and the Use Thereof in Breeding Programs | |
| CN108165653B (en) | InDel molecular marker for identifying pepper maturity and application thereof | |
| CN107488659B (en) | Sequence related to red and yellow color characters of citrus peels and application thereof | |
| CN113604596A (en) | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof | |
| CN108330201A (en) | Identify molecular labeling and its application of Tomato Mosaic Virus resistant gene | |
| CN109609687B (en) | KASP marker primer combination for detecting watermelon fusarium wilt resistance and application thereof | |
| CN111961751B (en) | KASP primer for detecting tomato root knot nematode resistance gene Mi-1.2 and application thereof | |
| CN110241245A (en) | Detect KASP primer and its application of cucumber bacterial angular leaf spot gene | |
| CN110607388A (en) | Wheat stripe rust resistant gene related SNP molecular marker in adult stage, primer and application thereof | |
| CN108796107B (en) | SNP molecular marker coseparated with cucumber spur hardness gene Hard and application thereof | |
| CN112195268B (en) | Molecular marker, primer, application and variety breeding method closely linked with origin green peach aphid resistance character of cultivar | |
| CN102747070A (en) | Two CAPs markers tightly linked with muskmelon anti-powdery mildew gene Pm-AN and its application method | |
| Sharma et al. | Genetic variability in strawberry (Fragaria x ananassa Duch.) cultivars assessed by morphological traits and EST-SSR markers of Rubus ellipticus | |
| CN113278723B (en) | Composition for analyzing genetic diversity of Chinese cabbage genome segment or genetic diversity introduced in synthetic mustard and application | |
| CN112011640B (en) | KASP molecular marker, primer and application for identifying pH of watermelon fruit | |
| CN116064904A (en) | Molecular marker closely linked with wheat stem rot resistance QTL Qfcr.sicau.2A and application | |
| KR20160095213A (en) | DNA marker for selecting fruit shape of watermelon | |
| CN114480721A (en) | Method for identifying whether melon variety to be detected is thin-skin melon or thick-skin melon and special SNP primer combination thereof | |
| CN103834647A (en) | SSR marker Xgwm537 in close linkage with wheat dwarf gene RhtDC20 and application thereof | |
| CN114525360A (en) | SNP marker closely linked with green fruit color Gv1 gene of eggplant and application | |
| CN108424954B (en) | Anthranilate synthase allele fragment capable of increasing rice yield and application thereof | |
| CN113481318B (en) | KASP primer for detecting powdery mildew disease-resistant gene mlo of cucumber and application thereof |
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 |