CN112391487B - SNP marker for identifying broccoli variety Zhe Qing 75 - Google Patents
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Abstract
The invention discloses an SNP marker for identifying a broccoli variety Zhe Qing 75', wherein bases of the SNP marker at the 42031808 position of the chromosome C1 of a broccoli HDEM reference genome, at the 6582159 position of the chromosome C6 of the broccoli HDEM reference genome and at the 38826789 position of the chromosome C7 of the broccoli HDEM reference genome are G or C respectively. Also provides a primer for detecting the SNP marker, and the nucleotide sequence of the primer is shown as SEQ ID NO. 1-9. The SNP marker and the primer thereof can identify the purity of the 'Zheqing 75' hybrid based on a KASP technical system, have stable and accurate results, and can identify the purity and the authenticity of the 'Zheqing 75' seed at high throughput.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and designs an SNP marker for identifying a broccoli variety Zhe Qing 75, in particular to an SNP marker for identifying the purity or authenticity of a broccoli variety Zhe Qing 75 developed based on a KASP technology.
Background
Broccoli (Brassica oleracea L.var. Italic), also known as broccoli, green cauliflower, etc., is one of Brassica brassicaceae Brassica varieties of vegetables of the cabbage family. Broccoli is highly popular with consumers both in and out due to its abundance as an anti-cancer active ingredient sulforaphane (Dinkova-Kostova A T, holtzclaw W D, cole R N, itoh K, wakabayashi N, katoh Y, yamamoto M, talalay P.2002.Direct evidence of sulfohydryl groups of Keap1 are the sensors regulating indication of phase 2enzymes present resistant genes and oxides. Proceedings of National Academy of Sciences USA,99 (18): 11908-11913). The demand and planting area of broccoli in China are increased year by year.
The broccoli variety Zhe Qing 75 is a high-quality medium-ripeness broccoli hybrid which is autonomously bred by vegetable research institute of agricultural academy of sciences in Zhejiang province. Harvested about 75 days after planting. Strong growth potential, moderate plant type and moderate branch number; the flower ball is mushroom-shaped, the spherical surface is flat, the flower stalk is long and green, the bud grains are fine and uniform, the bud color is dark green, the flower ball is not easy to purple at low temperature, and the marketability is good. Is suitable for planting in autumn and plateau areas in most areas of China.
The KASP technique (competitive allele specific PCR) is a new generation of high throughput automated SNP detection technique. The technology is highly flexible, so that the application of the technology is also quite wide (Wang Fujiang, fan Xiucai, zhang Ying, liu Chonghuai, jiang Jianfu. Application and prospect of SNP molecular markers in crop variety identification. Plant genetic resources declaration, DOI: 10.13430/j. Cnki. Jpgr.20200309002). The application provides technical support for realizing the genetic purity identification, the authenticity identification and the protection of the Zhejiang blue 75 variety by developing KASP labeled primers and establishing an identification method thereof.
Disclosure of Invention
The invention aims to provide an SNP marker based on KASP technology, which is used for identifying the purity and the authenticity of 'Zhe Qing 75' seeds, the method has stable and accurate results, and the purity and the authenticity of 'Zhe Qing 75' seeds can be identified in a high-throughput manner.
In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:
the invention obtains millions of SNP loci by utilizing the re-sequencing data of 23 parts of broccoli core germplasm, screens 100 SNP loci according to the Polymorphism Index (PIC) of each SNP locus, the Minimum Allele Frequency (MAF), the distribution on a chromosome and the like, and utilizes KASP technology to genotype 392 parts of broccoli material, thereby establishing broccoli fingerprint spectrum library data.
The invention determines 3 pairs of primer combinations based on the 'Zheqing 75' broccoli hybrid and the genotyping result of the parents thereof, and can be used for identifying the seed purity and authenticity of the 'Zheqing 75' hybrid.
In one aspect of the present invention, there is provided an SNP marker for identifying broccoli variety 'zhe qing 75', the SNP marker comprising:
a marker numbered SNP7501, wherein the base of the marker at the 42031808 position of the broccoli HDEM reference genome C1 chromosome is G or C;
a marker with the number of SNPSNP7501, wherein the base of the marker at the 6582159 position of the C6 chromosome of the broccoli HDEM reference genome is G or C;
a marker numbered SNPSNP7501 having a G or C base at position 38826789 of broccoli HDEM reference genome C7 chromosome.
In another aspect of the present invention, there is provided a primer set for detecting the above SNP marker, the primer set comprising:
the primer pair for detecting SNP7501 marker consists of a forward primer F1, a reverse primer H1 and a reverse primer C1, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1-3;
the primer pair for detecting SNP7502 markers consists of forward primers F2 and H2 and a reverse primer C2, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 4-6;
the primer pair for detecting SNP7503 marker consists of forward primers F3 and H3 and reverse primer C3, and the nucleotide sequence is shown in SEQ ID NO. 7-9.
Furthermore, the two forward primers in each labeled primer pair have base difference only at the 3' end, can be competitively combined with the target site, display corresponding FAM or HEX fluorescence, and can judge the genotype of the target site after signal amplification.
Based on KASP technical system, the purity of ' Zheqing 75' hybrid is identified, in the embodiment of the invention, 2 upstream primers are adopted, and universal adaptor sequences are respectively added at the 5' ends of the upstream primers. The sequence of the universal joint added by the upstream primers F1-3 is shown in SEQ ID NO. 10; the sequence of the universal joint added by the upstream primers H1-3 is shown in SEQ ID NO. 11.
Based on a KASP genotyping technology system, the SNP marker corresponding primers of the invention are as follows aiming at the genotypes of the broccoli 'Zhe Qing 75' and parents thereof:
in another aspect of the invention, the invention also provides a kit for identifying the broccoli variety Zhe Qing 75', wherein the kit comprises the primer combination shown in SEQ ID NO. 1-9.
Preferably, the kit also comprises a universal joint sequence shown as SEQ ID NO. 10-11.
In another aspect of the present invention, there is also provided a method for identifying broccoli variety 'Zhe Qing 75', comprising the steps of:
1) Extracting genomic DNA of broccoli to be detected;
2) Performing PCR amplification by using the primer combination and the universal joint;
3) And analyzing the PCR amplification product by using a fluorescence detector.
Further, the PCR reaction system is as follows: 5 mu L of PCR premix; 0.14 mu L of primer mixed solution, wherein the final concentration of each primer is 5nM;20 ng/. Mu.L template DNA 5. Mu.L.
Further, the PCR reaction program is as follows: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s, annealing and extension at 61-55 ℃ for 60s, wherein the annealing temperature of each cycle is reduced by 0.6 ℃, and the cycle time is 10; denaturation at 94 ℃ for 20s and annealing extension at 55 ℃ for 60s, and the cycle is 26.
Further, according to the genotyping result, the sample to be detected is judged to be a parent, a hybrid or a heterotypic strain.
The criteria for determination are as follows:
in each sample, if the genotype results of the 3 SNP markers are GG, GG and CC respectively, the sample can be judged as a male parent; if the genotype results are CC, CC and GG respectively, the sample can be judged as the female parent; if the genotype results are both GC, the sample can be judged as the 'Zheqing 75' hybrid F1; otherwise (except for the deletion genotype), the specimen is judged to be a heterotypic strain.
According to the judgment result, the number of the parents, the hybrids and the heterotypic strains is counted, and the seed purity of the Zheqing 75' can be calculated. 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 measured; p is the number of parent single plants; h is the number of the heterotypic plants; f is the number of hybrid seeds.
In another aspect of the invention, the application of the SNP marker or the primer combination in identifying the purity of the 'Zheqing 75' hybrid is also provided.
The invention has the beneficial effects that:
the invention screens 100 SNP loci based on 392 parts of broccoli representative material fingerprint data and a high-flux KASP genotyping platform, screens 3 SNP loci suitable for identifying the purity of 'Zhe Qing 75' seeds according to polymorphism indexes, minimum allele frequency, genotyping quality, result repeatability and the like, and designs 9 primer sequences for detecting the SNP loci. The 9 primer sequences can realize the high-flux and high-efficiency purity identification of the Zheqing 75' seeds. The invention also provides a method for judging the seeds as parents, hybrid seeds or heterotypic strains and calculating the purity, and provides a powerful technical support for the popularization of the broccoli variety Zhe Qing 75'.
Drawings
FIG. 1 is a diagram showing the typing effect of the specific primer set of SNP7501 site for identifying seed purity of Zheqing 75' on KASP technique platform according to the present invention; in the figure, the genotype of the cluster at the upper left corner is GG, the genotype of the cluster at the middle part is GC, and the genotype of the cluster at the lower right corner is CC;
FIG. 2 is a diagram showing the typing effect of the specific primer set of SNP7502 site for identifying seed purity of Zheqing 75' on KASP technique platform according to the present invention; in the figure, the genotype of the cluster at the upper left corner is GG, the genotype of the cluster at the middle part is GC, and the genotype of the cluster at the lower right corner is CC;
FIG. 3 is a diagram showing the typing effect of the specific primer set of SNP7503 site for identifying seed purity of Zheqing 75' on KASP technique platform according to the present invention; in the figure, the genotype of the cluster at the upper left corner is CC, the genotype of the cluster at the middle is CG, and the genotype of the cluster at the lower right corner is GG.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 SNP molecular markers and primer identification
1) Alternative sites: 100 SNP loci used for constructing 392 broccoli materials (provided by 11 broccoli breeding joint customs units in China) fingerprint maps.
2) And (3) site screening: from 100 SNP loci, 3 pairs of specific locus combinations with good repeatability and clear typing results are selected according to the polymorphic indexes of the loci, the minimum allele frequency and the genotyping results of the Zheqing 75 'hybrid and the parents thereof, and are used for identifying the purity of the Zheqing 75'.
3) The numbers of the 3 SNP markers are SNP7501, SNP7502 and SNP7503, respectively, and the site information thereof is as follows:
TABLE 1 SNP site information of broccoli variety Zhe Qing 75
4) The primer sequence for detecting the SNP sites based on the KASP technology determined by the invention and the PIC, MAF and heterozygosity rate information after genotyping 392 parts of materials are as follows:
TABLE 2 detection of SNP site information of broccoli based on KASP technique
Each pair of primers comprises 3 primer sequences, wherein F1-3 and H1-3 are respectively forward primers, and C1-3 are reverse primers. The two forward primers have base difference only at the 3' end and can be competitively combined with the target site to display corresponding FAM or HEX fluorescence, and the genotype of the target site can be judged after signal amplification.
Based on KASP technical system, the purity of ' Zheqing 75' hybrid is identified, in the embodiment of the invention, two upstream primers are adopted, and universal adaptor sequences are respectively added at the 5' ends of the upstream primers. The sequence of the universal joint added by the upstream primers F1-3 is 5'-GAAGGTGACCAAGT TCATGCT-3'; the sequence of the universal joint added by the upstream primers H1-3 is 5'-GAAGGTCGGA GTCAACGGATT-3'.
5) The genotype data of broccoli Zhe Qing 75' and its parents on the 3 pairs of SNP primers are as follows:
TABLE 3 genotype of Zhe Qing 75' and its parents
Example 2 identification of purity of the Zhejiang blue 75 broccoli hybrid
The embodiment specifically provides a method for identifying the purity of a 'Zhejiang cyan 75' broccoli hybrid, which comprises the following steps:
extraction of DNA
Randomly selecting 180 seeds of the 'Zheqing 75' hybrid seeds to be detected and 2 seeds of the parent and the parent respectively, and performing DNA preparation.
1) The seeds are sown in a plug tray, and after two weeks, 1 cm-sized leaves are put in a 2ml centrifuge tube, and glass beads with the diameter of 4mm are put in the centrifuge tube.
2) Adding 500ul of 2% CTAB, and fully grinding by a grinder;
3) After a water bath at 65 ℃ for 45min, an equal volume of chloroform-isoamyl alcohol (volume ratio 24: 1) And reversing and mixing evenly.
4) Centrifuge at 12000rpm for 15min and take the supernatant (approximately 400 ul) in a new 1.5ml tube.
5) Adding 2 times volume of absolute ethyl alcohol into the supernatant, and waiting for DNA precipitation.
6) Centrifuging at 10000rpm for 2min, pouring off supernatant, adding 75% ethanol, cleaning for 3 times, and air drying.
7) 200 μ l of TE (pH 8.0) or ddH was added 2 And O, fully dissolving for later use.
2. Genotyping detection Using the KASP technique
The purity of 'Zheqing 75' is identified by using a specific KASP marker consisting of markers SNP7501, SNP7502 and SNP 7503.
The method comprises the following specific steps:
1) Extracting the genomic DNA of the broccoli to be detected;
2) Adding specific KASP Primer mix and general KASP Master mix into the DNA template extracted in the step 1) for PCR amplification;
3) And analyzing the PCR amplification product by using a fluorescence detector.
The KASP Mastermix comprises the following components: universal FRET cassette fluorescent primer, ROX internal reference dye, klearTaq DNA polymerase, dNTP and MgCl 2 。
The PCR reaction system for KASP detection was as follows: 5 mu L of PCR premix; 0.14 mu L of primer mixed solution, wherein the final concentration of each primer is 5nM;20 ng/. Mu.L template DNA 5. Mu.L;
the reaction conditions for the PCR were: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for extension for 60s, and annealing temperature reduction of 0.6 ℃ in each cycle for 10 cycles; denaturation at 94 ℃ for 20s and annealing extension at 55 ℃ for 60s, and the cycle is 26.
3. Analysis of results
Through detection, 180 hybrid seeds 'Zhe Qing 75' to be detected and SNP marker genotyping results of the parents thereof with the numbers of SNP7501, SNP7502 and SNP7503 are obtained.
TABLE 4 KASP genotyping results for 180 samples tested
| Sample name | SNP7501 | SNP7502 | SNP7503 |
| Sample 1 | GC | GC | GC |
| Sample 2 | GC | GC | GC |
| Sample 3 | GC | GC | GC |
| …… | …… | …… | …… |
| …… | …… | …… | …… |
| Sample 179 | GC | GC | GC |
| Sample 180 | GC | GC | GC |
| NTC | Drop Out | Drop Out | Drop Out |
| P1 | GG | GG | CC |
| P2 | CC | CC | GG |
The statistical results show that: the genotyping results of 180 samples to be tested are GC by using 3 markers, the male parent is GG, GG and CC, the female parent is CC, CC and GG, and the purity of the hybrid is 100% (figure 1-3).
EXAMPLE 3 identification of Zhejiang blue 75
The broccoli seeds to be detected and the 'Zheqing 75' standard sample seeds are randomly selected for 48 seeds respectively, DNA is prepared according to the method in the embodiment 2, and 3 SNP markers related to the invention are utilized for genotyping detection. The PCR reaction system and reaction conditions were as in example 2.
TABLE 5 typing results of varieties to be tested and standard samples 'Zheqing 75' on 3 SNP sites
| Number of | Seed to be tested | Standard sample |
| SNP7501 | CC | GC |
| SNP7502 | GC | GC |
| SNP7503 | GG | GC |
And (4) analyzing results: through the detection, the typing results of the broccoli seeds to be detected and the Zhe cyan 75' at the 3 SNP sites are obtained, and the comparison analysis shows that: the difference between the hybrid to be detected and the Zheqing 75 'at the 2 SNP sites indicates that the hybrid to be detected is not the real hybrid of the Zheqing 75'.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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Claims (7)
1. A primer combination for identifying a broccoli variety 'Zhe Qing 75' is characterized by consisting of the following primers:
the nucleotide sequences of the forward primers F1 and H1 and the reverse primer C1 are shown in SEQ ID No. 1-3; the molecular marker of the primers F1, H1 and C1 is SNP7501, and the molecular marker SNP7501 has G/C polymorphism;
the nucleotide sequences of the forward primers F2 and H2 and the reverse primer C2 are shown in SEQ ID No. 4-6; the molecular marker of the primers F2, H2 and C2 is SNP7502, and the molecular marker SNP7502 has G/C polymorphism;
the nucleotide sequences of the forward primers F3 and H3 and the reverse primer C3 are shown in SEQ ID No. 7-9; the molecular marker amplified by the primers F3, H3 and C3 is SNP7503, and the molecular marker SNP753 has G/C polymorphism.
2. The primer combination of claim 1, wherein the 5 'end of the primers F1, F2, and F3 is connected to a universal linker having a sequence shown in SEQ ID NO.10, and the 5' end of the primers H1, H2, and H3 is connected to a universal linker having a sequence shown in SEQ ID NO. 11.
3. A kit for identifying a broccoli variety 'Zhe Qing 75', which comprises the primer combination shown in claim 1.
4. The kit according to claim 3, wherein the 5 'ends of the primers F1, F2 and F3 are connected with a universal linker having a sequence shown in SEQ ID No.10, and the 5' ends of the primers H1, H2 and H3 are connected with a universal linker having a sequence shown in SEQ ID No. 11.
5. A method for identifying a broccoli variety 'Zhe Qing 75' is characterized by comprising the following steps:
1) Extracting a broccoli genome DNA sample to be detected;
2) Performing PCR amplification using the primer combination of claim 1 and the universal adaptor of claim 2;
3) Analyzing the PCR amplification product by using a fluorescence detector; when the genotype results of the 3 molecular markers SNP7501, SNP7502 and SNP7503 are GG, GG and CC respectively, the sample can be judged as a male parent; when the genotype results of 3 molecular markers SNP7501, SNP7502 and SNP7503 are CC, CC and GG respectively, the sample can be judged as the female parent; when the genotype results of the 3 molecular markers SNP7501, SNP7502 and SNP7503 are all GC, the sample can be judged as the 'Zheqing 75' hybrid F1; the specimen can be judged as a heterotypic strain unless the genotype is deleted.
6. The method of claim 5, wherein the PCR reaction system is: 5 mu L of PCR premixed solution; 0.14 mu L of primer mixed solution, wherein the final concentration of each primer is 5nM;20 ng/. Mu.L template DNA 5. Mu.L.
7. The method of claim 5, wherein the PCR reaction is performed by: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s, annealing and extension at 61-55 ℃ for 60s, wherein the annealing temperature of each cycle is reduced by 0.6 ℃, and the cycle time is 10; denaturation at 94 ℃ for 20s and annealing extension at 55 ℃ for 60s, and the cycle is 26.
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