CN111961750A - KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof - Google Patents
KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof Download PDFInfo
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- 241000543828 Tomato yellow leaf curl Sardinia virus Species 0.000 claims description 5
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- 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
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- 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
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- 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 crop breeding, in particular to a KASP primer for detecting the genotyping of a tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof. The tomato yellow leaf curl virus disease resistance gene Ty-1 has an SNP site at the position of 3461 base, and the mutation of the base of the SNP site causes the resistance of the Ty-1 gene to be changed. The invention designs a group of KASP primers for genotyping and a kit containing the KASP primers based on the SNP locus. The KASP primer and the kit can be used for identifying whether the tomato contains the tomato yellow leaf curl virus disease resistance gene Ty-1 or not, and distinguishing the tomato containing the Ty-1 and resisting the tomato yellow leaf curl virus disease from the tomato without the Ty-1 tomato yellow leaf curl virus disease. Compared with the prior art, the method can be used for quickly, accurately and high-flux disease resistance identification of tomato plants in the seedling stage, reduces the workload of artificial inoculation and field disease resistance identification in the seedling stage, and can improve the tomato breeding efficiency, save the breeding cost and accelerate the breeding process.
Description
Technical Field
The invention relates to the technical field of molecular biology and crop breeding, in particular to a KASP primer for detecting a tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof.
Background
Tomato (Lycopersicon esculentum Mill.) is an annual or perennial herb of the genus Lycopersicon of the family Solanaceae, originates in south America, is a worldwide vegetable crop, and is one of the main cultivated vegetables in China. With the expansion of tomato cultivation area, the diversification of cultivation methods, continuous cropping obstacles and other influences, tomato plant diseases and insect pests are also aggravated continuously. In recent years, Tomato Yellow Leaf Curl Virus (TYLCV) caused by Tomato yellow leaf curl virus has become one of the most serious diseases in Tomato production worldwide. The upper leaves of the infected plants at the initial stage firstly show yellow flower leaves, the growth is slow or stopped, the internodes are shortened, and the plants are obviously dwarfed. When the disease is serious in the later period, the plant growth is stagnated and dwarfed, the fruit setting is difficult after the flowering, the fruit can not normally change color, and the loss reaches 100 percent.
The cultivation of new TYLCV resistant varieties is a fundamental way for preventing and treating the tomato diseases, the tomato is changed from traditional breeding to modern molecular marker-assisted breeding research along with the continuous progress of tomato breeding technology, the molecular marker-assisted selection is the selection of target characters on the DNA level, is not influenced by the environment, is not interfered by the obvious recessive relation of alleles, and has accurate and reliable selection results. At present, in BMT molecular test guidelines, the International Union of plant protection of New varieties (UPOV) has determined methods for constructing DNA molecular markers as SSR (Simple sequence repeats) and SNP (Single nucleotide polymorphism) markers. 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, develops a plurality of stable and efficient SNP typing technologies such as gene chips and competitive Allele Specific PCR (KASP) markers based on the SNP marker, and successfully replaces the traditional SNP electrophoretic analysis, wherein the KASP technology has the characteristics of high throughput, time saving and convenience, and is characterized in that the SNP is typed based on the Specific matching of the base at the tail end of a primer, the accurate double Allele judgment can be carried out on SNP loci in a wide genome DNA sample, and the SNP marker has high stability and accuracy and becomes the mainstream of SNP genotyping. 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.
At present, the tomato yellow leaf curl virus disease resistance genes mainly comprise: ty-1, Ty-2, Ty-3, Ty-4 and Ty-5. The Ty-1 and Ty-3/Ty3a genes in almost all disease-resistant variety breeding are considered as multiple alleles and are also the most important genes for resisting tomato yellow leaf curl virus, the disease resistance of the gene is durable and stable, and the gene is widely applied to tomato yellow leaf curl virus resistance breeding. Therefore, the KASP technology is utilized to quickly and accurately identify the disease-resistant gene Ty-1 and the like of the tomato yellow leaf curl virus disease, so that the breeding efficiency of new varieties can be greatly improved, and the breeding period can be shortened.
The KASP technique (competitive allele-specific PCR) is one of the currently mainstream genotyping methods in the world, and is based on the specific matching of the terminal bases of primers to perform accurate biallelic gene judgment on SNP and InDel at a specific site. The primers adopt 3 specific primers, wherein 2 upstream primers are adopted, the 3 'end is an allelic variant base, the 5' end is added with a universal fluorescent adaptor sequence, the downstream primers are common primers, and the conventional PCR amplification and the end-point method fluorescent signal detection are carried out.
The specific primer of KASP usually adopts 24bp-26bp, so that higher specificity can be realized, and the accuracy is higher compared with the conventional enzyme digestion electrophoresis; only conventional PCR amplification is needed, and the required time is short; expensive double-color labeled probes are not needed, and the flexibility is super-strong; minimal DNA sample requirements, no whole genome amplification; the kit is rapid and efficient, and can detect a few labels of a large number of samples in high throughput.
The method mainly comprises the following operation steps:
1. alleles-1 and-2 containing SNP sites as templates;
2. designing specific primers aiming at allele SNP sites: two forward primers and a universal reverse primer;
3. the 5' ends of the two forward primers are respectively connected with a specific detection primer sequence which can be combined with a fluorescent label;
4. extracting genome DNA of a sample to be detected as a template;
5. PCR amplification, fluorescent signal scanning of the amplified product and data analysis.
The KASP technique has the characteristics of flexibility, cheapness, and accuracy, and has been widely used in various fields. At present, no KASP marker has been found for use in the detection of tomato yellow leaf curl virus.
The applicant has not searched the patent literature of the subject matter related to the present application in the domestic patent database.
Disclosure of Invention
The invention aims to provide a KASP primer for detecting a tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof, can be used for identifying the tomato yellow leaf curl virus disease resistance gene Ty-1 and whether the tomato contains the tomato yellow leaf curl virus disease resistance gene Ty-1, distinguishing the tomato containing Ty-1 and resisting the tomato yellow leaf curl virus disease from the tomato without Ty-1 and sensing the tomato yellow leaf curl virus disease, distinguishing the homozygous tomato (Ty-1/Ty-1) resisting the tomato yellow leaf curl virus disease, the homozygous tomato (Ty-1/Ty-1) sensing the tomato yellow leaf curl virus disease and the heterozygous tomato (Ty-1/Ty-1) resisting the tomato yellow leaf curl virus disease, further breeding tomato varieties which contain Ty-1 genes and resist tomato yellow leaf curl virus diseases.
The overall technical concept of the invention is as follows:
a KASP primer for detecting a tomato yellow leaf curl virus disease resistance gene Ty-1, wherein the KASP primer sequence comprises:
forward primer Ty-1-F1: 5'-TCAATGAACCTCATCTCCATGT-3', the sequence of which is shown in SEQ No. 1;
forward primer Ty-1-F2: 5'-TCAATGAACCTCATCTCCATGC-3', the sequence of which is shown in SEQ No. 2;
the forward primer Ty-1-F1 and the forward primer Ty-1-F2 are respectively connected with different tag sequences.
The kit for identifying and detecting the tomato yellow leaf curl virus disease resistance gene Ty-1 comprises KASP primers which are respectively connected with forward primers Ty-1-F1 and forward primers Ty-1-F2 with different tag sequences; or a KASP primer comprising a forward primer Ty-1-F1 with a tag sequence A added to the 5 'end and a forward primer Ty-1-F2 with a tag sequence B added to the 5' end; or a KASP primer with the sequence of the forward primer 1 being 5'-GAAGGTGACCAAGTTCATGCTTCAATGAACCTCATCTCCATGT-3' and the sequence of the forward primer 2 being 5'-GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3'; or a KASP primer comprising the reverse primer sequence 5'-TGTTCCCATAAGATAAAAACTGTCA-3'.
The KASP primer or the kit is applied to the identification of tomato yellow leaf curl virus disease resistance gene Ty-1.
The KASP primer or the kit is applied to identifying whether tomato yellow leaf curl virus disease resistance gene Ty-1 is contained in tomato.
The application of the KASP primer or the kit in identifying or distinguishing the tomato containing Ty-1 and resisting the tomato yellow leaf curl virus disease from the tomato without Ty-1 and being infected with the tomato yellow leaf curl virus disease.
The KASP primer or kit can be used for distinguishing homozygous tomato yellow leaf curl virus disease resistant tomatoes (Ty-1/Ty-1), homozygous tomato yellow leaf curl virus disease sensitive tomatoes (Ty-1/Ty-1) and heterozygous tomato yellow leaf curl virus disease resistant tomatoes (Ty-1/Ty-1).
The KASP primer or the kit is applied to Ty-1 gene breeding of tomato TYLCV.
The specific technical concept of the invention is as follows:
the KASP primer sequence is as follows:
forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTTCAATGAACCTCATCTCCATGT-3', the sequence of which is shown in SEQ No. 3;
forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3', and the sequence is shown in SEQ No. 4.
The KASP primer sequence further comprises:
reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3', and its sequence is shown in SEQ No. 5.
The kit is characterized by further comprising:
tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 6;
a tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3', the sequence of which is shown in SEQ No. 7;
the two tag sequences are respectively connected with different fluorescent groups; the complementary sequences of the two tag sequences are connected with BHQ quenching genes; the label sequence A is connected with FAM fluorescent gene, and the label sequence B is connected with HEX fluorescent gene.
The application of the KASP primer or the kit in identifying the tomato yellow leaf curl virus disease resistance gene Ty-1 and identifying whether the tomato contains the tomato yellow leaf curl virus disease resistance gene Ty-1 is to amplify a tomato sample to be detected by using the kit and detect and analyze an amplification product.
The invention achieves the substantive characteristics and obvious technical progress that:
compared with the prior art, the KASP primer and the kit provided by the application can be used for quickly, accurately and high-flux disease resistance identification of tomato plants in the seedling stage, so that the workload of artificial inoculation and field disease resistance identification of breeders in the seedling stage is greatly reduced, the breeding efficiency can be improved, the breeding cost is saved, and the breeding process is accelerated. The KASP primer and the kit can be applied to the identification of tomato planting resources, various parents, hybrid seeds and other materials.
Drawings
FIG. 1 shows the results of typing tomato yellow leaf curl virus Ty-1 gene markers.
In the figure, the abscissa and ordinate values both represent fluorescence signal values, wherein the abscissa represents the HEX fluorescence signal value and the ordinate represents the FAM fluorescence signal value; the round point at the position I is an amplification signal of the tomato yellow leaf curl virus resistant material P808-1; round dots at II are amplification signals of the heterozygous tomato yellow leaf curl virus resistant material P808-1-P802-1-F1; the dot at the position III is an amplification signal of a tomato yellow leaf curl virus-infected material P802-1; the black dots at IV near the origin represent the amplification signal of the negative control (no DNA sample added).
FIG. 2 is the results of typing to identify tomato resistant to tomato yellow leaf curl virus and tomato susceptible to tomato yellow leaf curl virus.
In the figure, the abscissa and ordinate values both represent fluorescence signal values, wherein the abscissa represents the HEX fluorescence signal value and the ordinate represents the FAM fluorescence signal value; the round point at the position I is an amplification signal of a tomato yellow leaf curl virus resistant material; the round point at the position II is an amplification signal of the heterozygous tomato yellow leaf curl virus resistant material; the dot at the position III is an amplification signal of a tomato yellow leaf curl virus disease-sensitive material; the black dots at IV 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.
Tomato material used in the following examples: the tomato disease-resistant material P808-1, the tomato susceptible material P802-1 and the heterozygous tomato disease-resistant material P808-1-P802-1-F1 can be requested by the social public from the institute of economic crops of academy of agriculture and forestry, Hebei province 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 for identifying tomato yellow leaf curl virus disease resistance genes in high flux based on KASP technology, which comprises two specific amplification primers and a universal primer, and establishes a method for identifying the tomato yellow leaf curl virus disease resistance genes by applying a high flux molecular marker detection platform in combination with a Touchdown PCR method with strict application conditions.
Specifically, the applicant has found that there is a SNP site at the 3461 th base in the tomato yellow leaf curl virus disease resistance gene Ty-1 based on previous studies. The applicant verifies that the SNP site meets the requirement of KASP marker development (the genotype is different AT 3461 bp; no other SNP site exists nearby, the SNP site is located in a non-SNP dense region; and a sequence complex region with high continuous AT and GC contents is avoided). A large number of experiments are carried out on the 3461 th (T/C) SNP site of a tomato yellow leaf curl virus disease resistance gene Ty-1, and two specific forward primers and a universal reverse primer are screened and determined.
In specific embodiments, different forward or reverse primers are designed according to different needs of the base of the SNP site, for example, two forward primers are provided, including a forward primer Ty-1-F1: 5 'TCAATGAACCTCATCTCCATGT-3', and forward primer Ty-1-F2: 5'-TCAATGAACCTCATCTCCATGC-3', the last base of the two ends only at the 3 ' end is different, namely the corresponding SNP site; simultaneously, the 5' ends of the two forward primers are respectively connected with different label sequences, such as a label sequence A: GAAGGTGACCAAGTTCATGCT, and tag sequence B: GAAGGTCGGAGTCAACGGATT are provided.
For example, the present application provides a forward primer 1: 5' -GAAGGTGACCAAGTTCATGCTTCAATGAACCTCATCTCCATGT-3', and forward primer 2: 5' -GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3'. The underlined sequence is the added tag sequence.
In a specific embodiment, the KASP technique requires that the PCR amplification product is preferably 80bp-150bp in length, under which the sequence is variable. For example, reverse primers provided herein are: 5'-TGTTCCCATAAGATAAAAACTGTCA-3', the length of the amplified product is 110 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 the same as that of the tag sequence B, namely 5' -GAAGGTCGGAGTCAAC, GGATT-3 ', and 1 fluorescent group HEX is connected to the 5' end of the fluorescent probe B. 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. 8; the sequence of the quenching probe B is complementary with the label sequence B, is 5'-CTTCCAGCCTCAGTTGCCTAA-3', and has a sequence shown in SEQ No. 9; 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 the tomato yellow leaf curl virus disease resistance gene Ty-1 or the kit for detecting the tomato yellow leaf curl virus disease resistance gene Ty-1 provided by the application can be used for identifying whether tomato contains the tomato yellow leaf curl virus disease resistance gene Ty-1, can be used as a co-dominant marker for distinguishing homozygous tomato (Ty-1/Ty-1) resistant to tomato yellow leaf curl virus disease, homozygous tomato (Ty-1/Ty-1) susceptible to tomato yellow leaf curl virus disease and heterozygous tomato (Ty-1/Ty-1) resistant to tomato yellow leaf curl virus disease, and can accurately identify the genotype of Ty-1 so as to guide tomato breeding, prevention and control of tomato yellow leaf curl virus disease and the like.
In a specific embodiment, the present application provides a method for identifying whether tomato contains the disease resistance gene Ty-1 of tomato yellow leaf curl virus disease or not, which comprises amplifying DNA of a tomato sample to be identified using KASP primers provided herein, and detecting and analyzing the amplified product.
In particular embodiments, the KASP primers provided herein: for example, the forward primer 1: 5' -GAAGGTGACCAA GTTCATGCTTCAATGAACCTCATCTCCATGT-3', and forward primer 2: 5' -GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3', and reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3' are provided. If only FAM fluorescent signals are detected or FAM fluorescent signals and HEX fluorescent signals are detected simultaneously through amplification, the tomato sample to be identified is determined to contain the disease-resistant gene Ty-1 of the tomato yellow leaf curl virus; and (4) if only HEX fluorescent signals are detected, determining that the tomato sample to be identified does not contain the disease-resistant gene Ty-1 of the tomato yellow leaf curl virus disease.
In particular embodiments, the KASP primers provided herein: for example, the forward primer 1: 5' -GAAGGTGACCAA GTTCATGCTTCAATGAACCTCATCTCCATGT-3', and forward primer 2: 5' -GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3', and reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3' are provided. If only FAM fluorescent signals are detected by amplification, determining that the tomato sample to be detected contains homozygous tomato yellow leaf curl virus disease resistance gene Ty-1; if the FAM fluorescent signal and the HEX fluorescent signal are detected simultaneously by amplification, the tomato sample to be detected is determined to contain the heterozygous tomato yellow leaf curl virus disease resistance gene Ty-1; and (3) if only HEX fluorescent signals are detected, the tomato sample to be identified is determined to be free of the disease-resistant gene Ty-1 of the tomato yellow leaf curl virus.
In particular embodiments, the tomato sample to be identified may be taken from any of the leaves, roots, stems, flowers, fruits and seed species of a tomato plant.
In another embodiment, the present application provides the use of a method of differentiating between tomatoes which comprise a Ty-1 resistant tomato yellow leaf curl virus and tomatoes which do not comprise a Ty-1 sensitive tomato yellow leaf curl virus, and between homozygous tomato resistant to tomato yellow leaf curl virus (Ty-1/Ty-1), homozygous tomato resistant to tomato yellow leaf curl virus (Ty-1/Ty-1) and heterozygous tomato resistant to tomato yellow leaf curl virus (Ty-1/Ty-1), comprising amplifying DNA of a tomato sample to be identified using the KASP primers or kits provided herein, and detecting and analyzing the amplification products.
In particular embodiments, the KASP primers provided herein: for example, the forward primer 1: 5' -GAAGGTGACCAA GTTCATGCTTCAATGAACCTCATCTCCATGT-3', and forward primer 2: 5' -GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3', and reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3' are provided. If only FAM fluorescent signals are detected or FAM fluorescent signals and HEX fluorescent signals are detected simultaneously in the amplification, the tomato sample to be identified is determined to be tomato with tomato yellow leaf curl virus disease resistance; and (3) if only the HEX fluorescent signal is detected, the tomato sample to be identified is determined to be the tomato susceptible to tomato yellow leaf curl virus.
In particular embodiments, the KASP primers provided herein: for example, the forward primer 1: 5' -GAAGGTGACCAA GTTCATGCTTCAATGAACCTCATCTCCATGT-3', and forward primer 2: 5' -GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3', and reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3' are provided. If only FAM fluorescent signals are detected by amplification, the tomato sample to be detected is determined to be homozygous tomato yellow leaf curl virus disease resistant tomatoes; if the FAM fluorescent signal and the HEX fluorescent signal are detected simultaneously by amplification, the tomato sample to be detected is determined to be heterozygous tomato resistant to tomato yellow leaf curl virus; and (3) if only the HEX fluorescent signal is detected, the tomato sample to be identified is determined to be the tomato susceptible to tomato yellow leaf curl virus.
In particular embodiments, the tomato sample to be identified may be taken from any of the leaves, roots, stems, flowers, fruits and seed species of a tomato 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
Comparing and analyzing the sequences of the disease-resistant allele and the disease-susceptible allele of the Ty-1, and naming the disease-resistant allele of the Ty-1 as Ty-1/Ty-1 and the disease-susceptible allele of the Ty-1 as Ty-1/Ty-1; and one SNP site exists at the position of 3461 bp. The SNP site meets the requirement of KASP marker development: the genotypes are different at 3461 bp; the adjacent non-SNP locus is positioned in a non-SNP dense region; avoiding the complex sequence area with high continuous AT and GC contents.
Therefore, the T/C site at the 3461 th site is used as a unique selection target; genotype site T of Ty-1+ at this site, genotype site C of Ty-1.
Designing KASP primer according to the SNP locus, wherein the primer sequence is as follows:
3461 bit T/C:
forward primer 1: 5' -GAAGGTGACCAAGTTCATGCTTCAATGAACCTCATCTCCATGT-3’；
Forward primer 2: 5' -GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3’；
Wherein the underlined sequences are added tag sequences;
reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3' are provided.
The primer test results are shown in FIG. 1.
Example 2
Amplification of molecular markers
In the embodiment, the materials are used as high-generation homozygous disease-resistant materials P808-1(Ty-1/Ty-1) and susceptible materials P802-1(Ty-1/Ty-1) which are bred by the applicant, and are respectively expressed as disease resistance and disease susceptibility (the same applies below) through field identification, and the materials are hybridized to prepare the P808-1-P802-1-F1 heterozygous disease-resistant material (Ty-1/Ty-1) by taking the materials as parents. Leaf tissue of the plants was collected for genomic 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; forward primer 1: 0.15. mu.l (10 pmol/. mu.l); forward primer 2: 0.15. mu.l (10 pmol/. mu.l); reverse primer: 0.4. mu.l (10 pmol/. mu.l); and ddH 2O: 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 Biosystems7500Real-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 Biosystems7500Real-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 genotyping results are shown in FIG. 1. Wherein the dot at the position I is an amplification signal of the disease-resistant material P808-1, and only an FAM fluorescence signal is detected; the round point at the position II is an amplification signal of the hybrid disease-resistant material P808-1-P802-1-F1, and FAM fluorescence and HEX fluorescence are detected simultaneously; the dot at the position III is an amplification signal of the susceptible material P802-1, and only a HEX fluorescence signal is detected; the black dots at IV near the origin represent the amplification signal of the negative control (no DNA sample added). The amplification signals of two homozygous genotypes, namely the amplification signals of homozygous disease resistance and homozygous infection, are respectively connected with the origin, and the closer the included angle is to the right angle, the better the typing effect is.
The results show that the amplification signal for the KASP primer pair P808-1 is expected to be near the vertical axis, the amplification signal for P802-1 is expected to be near the horizontal axis, and the amplification signal for P808-1-P802-1-F1 is expected to be in an intermediate position,
the three genotypes can be obviously distinguished and clustered, and the group of primers can be used and successfully designed.
Example 4
Identifying and distinguishing tomato yellow leaf curl virus resistant tomato and tomato yellow leaf curl virus susceptible tomato by using the Ty-1 gene KASP primer or kit
In the embodiment, the materials are high-generation homozygous disease-resistant material P808-1, susceptible material P802-1 and P808-1-P802-1-F1 heterozygous disease-resistant material which is prepared by hybridizing the two materials serving as parents and is bred by the applicant. 253 parts of tomato planting resources are widely collected (the specific typing results are shown in table 1), and leaf tissues of plants are collected for extracting genome DNA.
By adopting the method described in the embodiment 2, 253 genome DNA samples of the materials are respectively extracted to be used as PCR amplification templates; performing PCR amplification using the KASP primer; PCR reactions were performed using an Applied Biosystems7500Real-Time PCR System; the PCR products were genotyped and data analyzed using the software available from the Applied Biosystems7500Real-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 detection typing results show that the number of individuals of the detection band type I in the same figure 2 is 84 (only FAM fluorescence signals are detected and the detection band type is homozygous disease-resistant materials), the number of individuals of the detection band type II in the same figure 2 is 73 (both FAM and HEX fluorescence signals are detected and the detection band type is heterozygous disease-resistant materials), the number of individuals of the detection band type III in the same figure 2 is 96 (only HEX fluorescence signals are detected and the detection band type is susceptible materials), and black dots of the detection band type IV in the same figure 2 close to the origin point represent amplification signals of a negative control (no DNA sample is added).
The results show that the designed primers can accurately identify the disease-resistant tomatoes and the disease-susceptible tomatoes.
Table 1 identification of typing results of tomato material against tomato yellow leaf curl virus disease
Sequence listing
<110> institute of economic crops of academy of agriculture, forestry and science of Hebei province
<120> KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 and application thereof
<130> none
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> an Artificial Sequence
<400> 1
tcaatgaacc tcatctccat gt 22
<210> 2
<211> 22
<212> DNA
<213> an Artificial Sequence
<400> 2
tcaatgaacc tcatctccat gc 22
<210> 3
<211> 43
<212> DNA
<213> an Artificial Sequence
<400> 3
gaaggtgacc aagttcatgc ttcaatgaac ctcatctcca tgt 43
<210> 4
<211> 43
<212> DNA
<213> an Artificial Sequence
<400> 4
gaaggtcgga gtcaacggat ttcaatgaac ctcatctcca tgc 43
<210> 5
<211> 25
<212> DNA
<213> an Artificial Sequence
<400> 5
tgttcccata agataaaaac tgtca 25
<210> 6
<211> 21
<212> DNA
<213> an Artificial Sequence
<400> 6
gaaggtgacc aagttcatgc t 21
<210> 7
<211> 21
<212> DNA
<213> an Artificial Sequence
<400> 7
gaaggtcgga gtcaacggat t 21
<210> 8
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 8
cttccactgg ttcaagtacg a 21
<210> 9
<211> 21
<212> DNA
<213> An Artificial Sequence
<400> 9
cttccagcct cagttgccta a 21
Claims (12)
1. The KASP primer for detecting the tomato yellow leaf curl virus disease resistance gene Ty-1 is characterized in that the KASP primer sequence comprises:
forward primer Ty-1-F1: 5'-TCAATGAACCTCATCTCCATGT-3', the sequence of which is shown in SEQ No. 1;
forward primer Ty-1-F2: 5'-TCAATGAACCTCATCTCCATGC-3', the sequence of which is shown in SEQ No. 2;
the forward primer Ty-1-F1 and the forward primer Ty-1-F2 are respectively connected with different tag sequences.
2. The KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 as claimed in claim 1, wherein when synthesizing the KASP primer, tag sequence A is added to 5' end of forward primer Ty-1-F1, and tag sequence A is: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 6; the 5' end of the forward primer Ty-1-F2 is added with a tag sequence B, wherein the tag sequence B is as follows: 5'-GAAGGTCGGAGTCAACGGATT-3', the sequence is shown in SEQ No. 7.
3. The KASP primer for detecting the tomato yellow leaf curl virus disease resistance gene Ty-1 as claimed in claim 2, wherein the KASP primer sequence is:
forward primer 1: 5'-GAAGGTGACCAAGTTCATGCTTCAATGAACCTCATCTCCATGT-3', the sequence of which is shown in SEQ No. 3;
forward primer 2: 5'-GAAGGTCGGAGTCAACGGATTTCAATGAACCTCATCTCCATGC-3', and the sequence is shown in SEQ No. 4.
4. The KASP primer for detecting tomato yellow leaf curl virus disease resistance gene Ty-1 as claimed in any one of claims 1 to 3, wherein said KASP primer sequence further comprises:
reverse primer: 5'-TGTTCCCATAAGATAAAAACTGTCA-3', and the sequence is shown in SEQ No. 5.
5. Kit for identifying and detecting the tomato yellow leaf curl disease resistance gene Ty-1, characterized in that it comprises the KASP primer of any one of claims 1 to 4.
6. The kit according to claim 5, further comprising:
tag sequence A: 5'-GAAGGTGACCAAGTTCATGCT-3', the sequence of which is shown in SEQ No. 6;
a tag sequence B: 5'-GAAGGTCGGAGTCAACGGATT-3', the sequence of which is shown in SEQ No. 7;
the two tag sequences are respectively connected with different fluorescent groups; the complementary sequences of the two tag sequences are connected with BHQ quenching genes; the label sequence A is connected with FAM fluorescent gene, and the label sequence B is connected with HEX fluorescent gene.
7. Use of the KASP primer according to any one of claims 1 to 4 or the kit according to claims 5 to 6 for identifying the tomato yellow leaf curl virus disease resistance gene Ty-1.
8. Use of a KASP primer according to any one of claims 1 to 4 or a kit according to claims 5 to 6 for identifying whether tomato contains the tomato yellow leaf curl virus disease resistance gene Ty-1 in tomato.
9. Use of a KASP primer according to any one of claims 1 to 4 or a kit according to claims 5 to 6 to identify or distinguish between tomatoes which contain Ty-1 and are resistant to tomato yellow leaf curl virus disease and tomatoes which do not contain Ty-1 and are susceptible to tomato yellow leaf curl virus disease.
10. Use of a KASP primer according to any of claims 1 to 4 or a kit according to claims 5 to 6 for differentiating between homozygous tomato yellow leaf curl disease resistant tomatoes (Ty-1/Ty-1), homozygous tomato yellow leaf curl disease sensitive tomatoes (Ty-1/Ty-1) and heterozygous tomato yellow leaf curl disease resistant tomatoes (Ty-1/Ty-1).
11. Use of a KASP primer according to any one of claims 1 to 4 or a kit according to claims 5 to 6 for breeding a Ty-1 gene for resistance to TYLCV in tomato.
12. Use according to any one of claims 8 or 9, characterized in that the tomato sample to be detected is amplified using a kit, and the amplification products are detected and analyzed.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337636A (en) * | 2021-07-22 | 2021-09-03 | 中国农业科学院蔬菜花卉研究所 | SNP (Single nucleotide polymorphism) site and KASP (Kaposi-phosphate) molecular marker for identifying leaf vein traits of tomato and application |
| CN113604596A (en) * | 2021-08-10 | 2021-11-05 | 河北省农林科学院经济作物研究所 | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof |
| CN113736866A (en) * | 2021-09-30 | 2021-12-03 | 中国农业科学院农业基因组研究所 | SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof |
-
2020
- 2020-09-14 CN CN202010962630.2A patent/CN111961750A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337636A (en) * | 2021-07-22 | 2021-09-03 | 中国农业科学院蔬菜花卉研究所 | SNP (Single nucleotide polymorphism) site and KASP (Kaposi-phosphate) molecular marker for identifying leaf vein traits of tomato and application |
| CN113604596A (en) * | 2021-08-10 | 2021-11-05 | 河北省农林科学院经济作物研究所 | KASP primer for detecting cucumber small zucchini yellow mosaic virus disease resistance gene zym and application thereof |
| CN113736866A (en) * | 2021-09-30 | 2021-12-03 | 中国农业科学院农业基因组研究所 | SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof |
| CN113736866B (en) * | 2021-09-30 | 2022-06-24 | 中国农业科学院农业基因组研究所 | SNP locus combination for detecting tomato yellow leaf curl virus resistance and application thereof |
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