CN117568520A - SNP molecular marker primer set for identifying purity of Cauliflower hybrid of 'purple nepheline 65' and application thereof - Google Patents
SNP molecular marker primer set for identifying purity of Cauliflower hybrid of 'purple nepheline 65' and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker primer set for identifying purity of a Cauliflower hybrid of 'Cauliflower 65' and application thereof, wherein the primer set comprises: 3 SNP marker primer sets respectively marked as an S011 primer set, an S012 primer set and an S026 primer set; wherein, in the S011 primer group, the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 1-3; in the S012 primer group, the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 4-6; in the S026 primer group, the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 7-9. The invention can realize high-flux and high-efficiency purity identification of seeds of ' purple Cabernet sauvignon 65', provides powerful technical support for popularization of purple Cabernet sauvignon 65' and provides technical support for realizing genetic purity identification, authenticity identification and protection of the purple Cabernet sauvignon.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a SNP molecular marker primer set for identifying purity of a Cauliflower hybrid of 'Cauliflower 65' and application thereof.
Background
Broccoli (Brassica oleracea l. Var. Botrytis) is a variant of the brassica species of the cruciferae family, originating on the coast of the Mediterranean sea, and introduced into our country in the nineteenth century. At present, china is a country with large cauliflower cultivation area in the world. However, in the production, the cultivated broccoli varieties in China mainly comprise 'white jade 80', 'S100', and the like, and the varieties are single. Along with the improvement of the living standard of people, the requirement for the broccoli variety is diversified. Therefore, the cultivation of the new color cauliflower variety with independent intellectual property rights has important significance for promoting the development of the cauliflower industry in China and meeting the increasing living demands of people.
In recent years, a series of novel broccoli varieties are bred by a broccoli breeder in China through a plurality of years of efforts. The development of a rapid and accurate variety identification method lays a foundation for genetic purity analysis, identification of variety authenticity and protection of independent intellectual property rights of new varieties, and has important practical value. Traditional identification methods are investigated by field phenotype observation, are time-consuming, labor-consuming and occupied, and are limited by seasons. In recent years, methods for rapid and accurate identification of a variety of species have been developed using modern molecular biology techniques, such as KASP (KompetitiveAllele Specific PCR, competitive allele-specific PCR).
'Violet 65' is a high quality early maturing purple broccoli hybrid autonomously selected by vegetable institute of agricultural sciences, zhejiang province. The new plant variety protection of the country is applied for 5 months in 2023. 'Zixia 65' was harvested approximately 65 days after fixation. The plant grows fast, the plant is strong and neat, the flower ball is half-loose, the spherical bud grain is fine, the purple red color is formed, and the anthocyanin is rich; the peduncles are light green, have moderate length and are crisp and tender in taste. To ensure seed purity of the 'purple nepheline 65' hybrid and to normalize the purple cauliflower hybrid seed industry, an accurate and stable detection method was developed for genetic purity, variety identity and authenticity identification.
Disclosure of Invention
The invention aims to provide a SNP molecular marker primer set for identifying the purity of a Cabernet ' Cabernet 65' hybrid and application thereof, which can realize high-throughput and high-efficiency purity identification of a Cabernet ' seed, provide powerful technical support for popularization of a Cabernet ' Cabernet 65' variety and provide technical support for identification of genetic purity, authenticity identification and protection of the Cabernet variety.
In order to achieve the above object, the present invention provides a SNP molecular marker primer set for identifying purity of a Cauliflower hybrid of 'Cauliflower 65', comprising: 3 SNP marker primer sets respectively marked as an S011 primer set, an S012 primer set and an S026 primer set; in the S011 primer group, the nucleotide sequences of two forward primers are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 3; in the S012 primer group, the nucleotide sequences of two forward primers are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 6; in the S026 primer group, the nucleotide sequences of two forward primers are respectively shown as SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 9.
Preferably, FAM fluorescent markers are attached to the forward primers shown as SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO. 7; HEX fluorescent markers are connected to the forward primers shown in SEQ ID No.2, SEQ ID No.5 and SEQ ID No. 8.
Another object of the present invention is to provide a KASP kit comprising the SNP molecular marker primer set as described.
Another object of the present invention is to provide a gene chip of the SNP molecular marker primer set as described.
The invention also aims to provide the application of the SNP molecular marker primer set or the KASP reagent detection kit or the gene chip in the identification of the purity or the seed authenticity of the Cauliflower hybrid of 'Cabernet sauvignon 65'.
It is another object of the present invention to provide a method for identifying purity or seed authenticity of a 'Cabernet 65' cauliflower hybrid, the method comprising the steps of:
(1) Extracting the genome DNA of the cauliflower to be detected;
(2) Adding a specific KASP primer mixture and a universal KASP Mastermix into the DNA template extracted in the step (1) for PCR amplification; wherein the specific KASP primer mixed solution is the SNP molecular marker primer set as set forth in claim 1 or 2;
(3) Analyzing PCR amplified products by a fluorescence detector, and judging whether a sample to be detected is a parent, a hybrid or a heteromorphic strain according to a genotyping result, wherein the judgment standard is as follows:
for each sample, if the genotype results of the 3 SNP markers are consistent with the genotypes of the male parent or the female parent, judging that the sample is the male parent or the female parent; if the genotype results of the 3 SNP markers are heterozygous for the parental genotype, judging that the sample is a hybrid of 'purple nepheline 65'; if genotypes of the father parent and the hybrid of 'purple nepheline 65' appear simultaneously in the genotype results of the 3 SNP markers, the sample is judged to be a heterogenic strain.
Preferably, the universal KASP Mastermix comprises: universal FRET cassette fluorescent primer, ROX reference dye, klearTaq DNA polymerase, dNTP and MgCl 2 。
Preferably, the reaction system of the PCR is as follows: mu.L of universal KASP Mastermix, 0.14. Mu.L of specific KASP primer mix and 5. Mu.L of 20 ng/. Mu.L of template DNA; wherein, the final concentration of each primer is 5nM.
Preferably, the reaction conditions of the PCR are: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles; denaturation at 94℃for 20s and annealing at 55℃for 60s for 26 cycles.
Preferably, the calculation formula of the purity of the broccoli hybrid of 'purple nepheline 65' is as follows:
Pur(％)＝[(N-P-H)/F]×100％
wherein Pur is seed purity; n is the number of samples to be tested; p is the number of single plants of the parent; h is the abnormal plant number; f is the number of hybrid seeds.
The SNP molecular marker primer set for identifying the purity of the Cauliflower hybrid of 'Cauliflower 65' and the application thereof have the following advantages:
based on fingerprint spectrum data of a representative material of early 236 parts of broccoli, 100 SNP loci are screened based on a high-throughput KASP genotyping platform, 3 SNP loci suitable for purity identification of seeds of 'purple nepheline 65' are screened according to polymorphism indexes, minimum allele frequencies, quality of genotyping, repeatability of results and the like, and 9 primer sequences for detecting the SNP loci are designed. By using the 9 primer sequences, the high-flux and high-efficiency purity identification of the seed of 'purple nepheline 65' can be realized. The invention also provides a method for judging the seeds as parents, hybrid seeds or abnormal strains and calculating the purity, which provides powerful technical support for the popularization of the purple cauliflower variety 'purple nepheline 65' and provides technical support for the realization of the identification of the genetic purity, the identification of the authenticity and the protection of the purple cauliflower variety.
Drawings
FIG. 1 is a graph of the typing effect of a specific primer set at the S011 site on the KASP technical platform for identification of seed purity of 'Zixia 65'; in the figure, the genotype of the upper left cluster is AA, the genotype of the middle cluster is AT, and the genotype of the lower right cluster is TT.
FIG. 2 is a graph showing the effect of typing of a specific primer set for the S012 site on the KASP technical platform for identification of seed purity of 'Zixia 65'; in the figure, the genotype of the upper left cluster is CC, the genotype of the middle cluster is GC, and the genotype of the lower right cluster is GG.
FIG. 3 is a graph showing the effect of typing of a specific primer set for the S026 site on the KASP technical platform for identification of seed purity of 'Zixia 65'; in the figure, the genotype of the upper left cluster is CC, the genotype of the middle cluster is GC, and the genotype of the lower right cluster is GG.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following examples are given to illustrate the invention and, unless otherwise indicated, are set forth under conventional experimental conditions.
The KASP Master mix used in the examples below was purchased from LGC company, UK.
Example 1 determination of SNP molecular markers and primers for identification of purity of the' Cabernet Cavictoriae hybrid
(1) Alternative sites: a large number of SNP loci are obtained by utilizing the resequencing data of 19 parts of broccoli core germplasm, 100 SNP loci are screened according to Polymorphism Indexes (PICs), minimum Allele Frequencies (MAFs), distribution on chromosomes and the like of each SNP locus, 236 parts of broccoli material are genotyped by utilizing a KASP technology, and the broccoli fingerprint database data is established.
(2) Screening of sites: based on a high-throughput KASP genotyping platform, 3 SNP locus combinations suitable for purity identification of 'purple nepheline 65' seeds are screened out from 100 SNP loci according to polymorphism indexes of loci, minimum allele frequency, quality of genotyping, result repeatability and the like, and the 3 SNP locus combinations selected by the high-throughput KASP genotyping platform have good repeatability and clear typing result and can be used for purity identification of 'purple nepheline 65'.
(3) The numbers of 3 SNP markers are S0011, S012 and S026, respectively, and their site information is as follows:
TABLE 1 SNP site information of the broccoli variety' Cabernet 65
(4) The primer sequence for detecting the SNP locus based on the KASP technology, which is determined by the invention, has the following information:
TABLE 2KASP primer info
Each set of primers included F (FAM), H (HEX) and C (Common) 3 primer sequences, where F and H are two forward primers and C is the reverse primer. The F and H primers only have base difference at the 3' -end, can be competitively combined with a target site, display corresponding FAM or HEX fluorescence, and can judge the genotype of the target site after signal amplification.
(5) The genotype data of cauliflower 'purple nepheline 65' and its parents on 3 pairs of SNP primers are as follows:
example 2 method for identifying purity of Cauliflower hybrid of 'Cabernet sauvignon 65' Using SNP markers provided by the invention
1. Extraction of DNA
Randomly selecting 180 seeds of the 'purple nepheline 65' hybrid seeds and 2 seeds of the parent and the male parent to be detected, and extracting DNA, wherein the specific process is as follows:
(1) Sowing seeds in a culture dish, taking cotyledons after one week in a 2mL centrifuge tube, and putting glass beads with the diameter of 4 mm;
(2) 500. Mu.L of 2% CTAB was added and the mixture was sufficiently ground with a grinder;
(3) After water bath at 65 ℃ for 45min, adding equal volume of chloroform-isoamyl alcohol (volume ratio is 24:1), and mixing uniformly;
(4) Centrifuging at 12000rpm for 15min, and collecting about 400 μl of supernatant in a new 1.5mL tube;
(5) Adding 2 times of absolute ethyl alcohol into the supernatant, and uniformly mixing;
(6) Centrifuging at 10000rpm for 2min, removing supernatant, adding 75% ethanol to wash DNA precipitate for 3 times, and air drying;
(7) 200. Mu.L ddH was added 2 O is fully dissolved and is placed in a refrigerator at 4 ℃ for standby.
2. Genotyping assays using the KASP technique
The KASP reaction test was performed on an IntelliQube high throughput genotyping platform (LGC, biosearch Technologies). Purity was identified for 'purple nepheline 65' using a specific KASP marker consisting of markers S011, S012 and S026.
The PCR reaction system for KASP detection was as follows: mu.L of PCR premix, 0.14. Mu.L of primer mix and 5. Mu.L of 20 ng/. Mu.L of template DNA. Wherein, the final concentration of each primer is 5nM.
The PCR premix (KASP Mastermix) contained: universal FRET cassette fluorescent primer, ROX reference dye, klearTaq DNA polymerase, dNTP and MgCl 2 。
The reaction conditions for PCR for KASP detection were: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles; denaturation at 94℃for 20s and annealing at 55℃for 60s for 26 cycles.
After the reaction is completed, the fluorescence data of KASP reaction products are read by utilizing IntelliQube, and the result of fluorescence scanning is automatically converted into a graph.
3. Analysis of results
According to genotyping results, judging whether the sample to be detected is a parent, a hybrid or a heterogenic strain, wherein the judgment standards are as follows: for each sample, if the genotype results of the 3 SNP markers are consistent with the genotypes of the male parent or the female parent, judging that the sample is the male parent or the female parent; if the genotype results of the 3 SNP markers are heterozygous for the parental genotype, the sample can be judged to be a hybrid F1 of 'purple nepheline 65'; if the genotypes of the parent and F1 appear simultaneously in the genotype results of the 3 SNP markers, the sample can be judged to be a heterotype strain (i.e., a hybrid strain).
According to the judging result, counting the numbers of parents, hybrid seeds and abnormal strains, and calculating the seed purity of 'purple nepheline 65'. The purity calculation formula is as follows:
Pur(％)＝[(N-P-H)/F]×100％
wherein Pur is seed purity; n is the number of samples to be tested; p is the number of single plants of the parent; h is the abnormal plant number; f is the number of hybrid seeds.
Through detection, SNP marker genotyping results of 180 seeds of the hybrid seed 'purple nepheline 65' to be detected and parents thereof with the numbers of S011, S012 and S026 are obtained. Statistical results show that: the 180 samples to be tested are heterozygous by using 3 marking gene typing results, and the purity of the hybrid is 100%.
Example 3 identification of the broccoli to be tested Using SNP markers as a hybrid `Violet 65'
The cauliflower seeds to be tested (50 days of Pinus thunbergii, 70 days of Pinus thunbergii and 88 days of Pinus thunbergii) and 48 seeds of the standard sample seed of 'Zixia 65' were randomly selected, DNA was extracted according to the method of example 2, and genotyping was performed using the 3 SNP markers according to the present invention, and the PCR reaction system and the reaction conditions were as in example 2.
Analysis of results: through the detection, the typing result of the cauliflower seeds to be detected and the 'purple nepheline 65' on 3 SNP loci is obtained, and comparison analysis shows that the hybrid seeds to be detected have differences between 2 SNP loci and the 'purple nepheline 65', and the hybrid seeds to be detected are not true hybrid seeds of the 'purple nepheline 65'. The SNP marker can identify 'purple nepheline 65' from Thunberg pine flowers for 50 days, thunberg pine flowers for 70 days and Thunberg pine flowers for 88 days.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (10)
1. A SNP molecular marker primer set for identifying purity of a 'eucrypti 65' cauliflower hybrid, the primer set comprising: 3 SNP marker primer sets respectively marked as an S011 primer set, an S012 primer set and an S026 primer set;
in the S011 primer group, the nucleotide sequences of two forward primers are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 3;
in the S012 primer group, the nucleotide sequences of two forward primers are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 6;
in the S026 primer group, the nucleotide sequences of two forward primers are respectively shown as SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 9.
2. The SNP molecular marker primer set according to claim 1, wherein FAM fluorescent markers are attached to the forward primers shown as SEQ ID NO.1, SEQ ID NO.4 and SEQ ID NO. 7; HEX fluorescent markers are connected to the forward primers shown in SEQ ID No.2, SEQ ID No.5 and SEQ ID No. 8.
3. A KASP kit comprising the SNP molecular marker primer set according to claim 1 or 2.
4. A gene chip comprising the SNP molecular marker primer set according to claim 1 or 2.
5. Use of the SNP molecular marker primer set of claim 1 or 2 or the KASP kit of claim 3 or the gene chip of claim 4 for identifying purity or seed authenticity of a 'eucrypti 65' cauliflower hybrid.
6. A method for identifying purity or seed authenticity of a 'kale 65' cauliflower hybrid, the method comprising the steps of:
(1) Extracting the genome DNA of the cauliflower to be detected;
(2) Adding a specific KASP primer mixture and a universal KASP Master mix into the DNA template extracted in the step (1) for PCR amplification; wherein the specific KASP primer mixed solution is the SNP molecular marker primer set as set forth in claim 1 or 2;
(3) Analyzing PCR amplified products by a fluorescence detector, and judging whether a sample to be detected is a parent, a hybrid or a heteromorphic strain according to a genotyping result, wherein the judgment standard is as follows:
for each sample, if the genotype results of the 3 SNP markers are consistent with the genotypes of the male parent or the female parent, judging that the sample is the male parent or the female parent; if the genotype results of the 3 SNP markers are heterozygous for the parental genotype, judging that the sample is a hybrid of 'purple nepheline 65'; if genotypes of the father parent and the hybrid of 'purple nepheline 65' appear simultaneously in the genotype results of the 3 SNP markers, the sample is judged to be a heterogenic strain.
7. The method of claim 6, wherein the universal KASP Master mix comprises: universal FRET cassette fluorescent primer, ROX reference dye, klear Taq DNA polymerase, dNTP and MgCl 2 。
8. The method of claim 6, wherein the reaction system of the PCR is: mu.L of universal KASP Master mix, 0.14. Mu.L of specific KASP primer mix and 5. Mu.L of 20 ng/. Mu.L of template DNA; wherein, the final concentration of each primer is 5nM.
9. The method of claim 6, wherein the reaction conditions of the PCR are: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles; denaturation at 94℃for 20s and annealing at 55℃for 60s for 26 cycles.
10. The method of claim 6, wherein the purity of the broccoli hybrid of "sauerkraut 65" is calculated as:
Pur(％)＝[(N-P-H)/F]×100％
wherein Pur is seed purity; n is the number of samples to be tested; p is the number of single plants of the parent; h is the abnormal plant number; f is the number of hybrid seeds.
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