CN112029898B

CN112029898B - SNP marker for identifying broccoli variety Zhe Qing 100

Info

Publication number: CN112029898B
Application number: CN202011133432.1A
Authority: CN
Inventors: 沈钰森; 顾宏辉; 王建升; 虞慧芳; 赵振卿; 盛小光
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2022-10-18
Anticipated expiration: 2040-10-21
Also published as: CN112029898A

Abstract

The invention discloses an SNP marker for identifying a broccoli variety 'Zhe Qing 100', wherein the base of the SNP marker at the position of 20246816 of a chromosome C3 of a broccoli HDEM reference genome is T or C, the base at the position of 51751432 of a chromosome C4 of the broccoli HDEM reference genome is G or A, and the base at the position of 54974753 of a chromosome C7 of the broccoli HDEM reference genome is A or G. 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 100' hybrid based on a KASP technical system, have stable and accurate results, and can identify the purity and the authenticity of the 'Zheqing 100' seed in high throughput.

Description

SNP marker for identifying broccoli variety Zhe Qing 100

Technical Field

The invention belongs to the technical field of molecular biology, and designs an SNP marker for identifying a broccoli variety Zhe Qing 100', in particular to an SNP marker for identifying the purity or authenticity of the broccoli variety Zhe Qing 100 developed based on a KASP technology.

Background

Broccoli (Brassica oleracea L. Var. Italic), also known as cauliflower, broccoli, cauliflower, etc., is one of the cabbage vegetable varieties originating from coastal areas of the European Mediterranean region. Broccoli is highly favored by domestic and foreign consumers for its abundance of sulforaphane, an active anticancer ingredient (Dinkova-Kostova A T, holtzclaw W D, cole R N, itoh K, wakabayashi N, katoh Y, yamamoto M, talalay P.2002.Direct evidence of which Sulfhydryl groups of the Keap1 arm the sensing and regulating of phase 2 enzymes of which protection against pathogens and oxidation, proceedings of the National Academy of Sciences USA,99 (18): 11908-11913). The demand and planting area of broccoli are also increasing year by year. However, in the history of over 30 years of broccoli cultivation in China, the production seeds mainly depend on foreign import. Although domestic breeders strive for many years to breed some excellent new broccoli varieties. However, more than 90% of broccoli seeds are still imported in the current production, the seed price is high, and the method is extremely unfavorable for the benign healthy cycle development of broccoli industry. Therefore, the cultivation and popularization of the excellent broccoli variety with independent knowledge have important significance for the development of national economy and the healthy development of broccoli industry.

The broccoli variety Zhe Qing 100 is a high-quality late-maturing broccoli hybrid which is autonomously bred by vegetable research institute of agricultural academy of sciences in Zhejiang province. Harvested about 100 days after planting, strong growth potential, medium plant type and few branches; the ball flower is compact and round, the bud grains are fine and uniform, the bud color is dark green, the bud is not easy to be purple at low temperature, and the commodity is good. Is suitable for planting in Yangtze river basin and Yunnan autumn and winter.

The genetic purity identification, the authenticity identification and the protection of new varieties urgently need an efficient, rapid, accurate, economical and practical variety identification method. The SNP marker (Single Nucleotide Polymorphism) was recommended by the International plant protection Association (UPOV) in 2010 as one of the methods for crop variety identification and fingerprint databases. As the SNP has the advantages of rich polymorphism, easy genotyping, high genetic stability and the like, the SNP is considered to be one of the genetic markers with the best application prospect. The KASP (competitive allele Specific PCR) technique is based on the Specific matching of primer bases to type SNPs, can perform accurate biallelic judgment on SNP sites in large sample genomic DNA, and has the characteristics of high stability and accuracy. The application provides technical support for realizing the genetic purity identification, authenticity identification and protection of the 'Zheqing 100' 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 authenticity of 'Zhe Qing 100' seeds, the method has stable and accurate results, and the purity and authenticity of 'Zhe Qing 100' seeds can be identified at high throughput.

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 genotyping results of the broccoli hybrid seeds 'Zhe Qing 100' and parents thereof, and can be used for identifying the seed purity and authenticity of the 'Zhe Qing 100' hybrid seeds.

In one aspect of the present invention, there is provided an SNP marker for identifying broccoli variety 'zhe qing 100', the SNP marker comprising:

a marker of number KCM1001, the marker having a base at position 20246816 of the broccoli HDEM reference genome C3 chromosome as T or C;

a marker of number KCM1002, the marker having a base G or a at position 51751432 of the broccoli HDEM reference genome C4 chromosome;

a marker numbered KCM1003 having a base at position 54974753 of the C7 chromosome of the broccoli HDEM reference genome which is a or G.

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 the KCM1001 marker consists of forward primers F1 and 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 the KCM1002 marker 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 the KCM1003 marker consists of forward primers F3 and H3 and a reverse primer C3, and the nucleotide sequence of the primer pair is shown as 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 100' 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 the KASP genotyping technology system, the SNP marker corresponding primers of the invention are as follows for the genotypes of the broccoli 'Zhe Qing 100' and its parents:

sample name	KCM1001	KCM1002	KCM1003
				Zheqing 100	TC	GA	AG
Male parent	TT	GG	AA
				Female parent	CC	AA	GG

In another aspect of the invention, the invention also provides a kit for identifying the broccoli variety 'Zhe Qing 100', 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 100', comprising the steps of:

1) Extracting the genomic DNA of the 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: 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 3 SNP markers are TT, GG and AA respectively, the sample can be judged as a male parent; if the genotype results are respectively CC, AA and GG, the sample can be judged as the female parent; if the genotype results are TC, GA and AG respectively, the sample can be judged as the Zheqing 100 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 parents, hybrid seeds and heterotypic strains is counted, and the seed purity of 'Zheqing 100' 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 hybrids.

In another aspect of the invention, the application of the SNP marker or the primer combination in identifying the purity of the 'Zheqing 100' 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 purity identification of Zhe Qing 100' seeds according to polymorphism index, 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 be used for realizing the high-throughput and high-efficiency purity identification of the 'Zheqing 100' 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 100'.

Drawings

FIG. 1 is a diagram showing the typing effect of the specific primer set of KCM1001 locus for seed purity identification of Zheqing 100' on KASP technique platform according to the present invention; in the figure, the genotype of the upper left cluster is TT, the genotype of the middle cluster is TC, and the genotype of the lower right cluster is CC;

FIG. 2 is a diagram showing the typing effect of the specific primer set of KCM1002 locus for identifying seed purity of 'Zheqing 100' in the 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 GA, and the genotype of the cluster at the lower right corner is AA;

FIG. 3 is a chart of the typing effect of the specific primer set of KCM1003 locus used for purity identification of 'Zheqing 100' seeds on KASP technical platform according to the present invention; in the figure, the genotype of the upper left cluster is AA, the genotype of the middle cluster is AG, and the genotype of the lower right cluster 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 obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1SNP 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 index of the loci, the minimum allele frequency and the genotyping results of the Zheqing 100 'hybrid and the parents thereof, and are used for identifying the purity of the Zheqing 100'.

3) The numbers of the 3 SNP markers are KCM1001, KCM1002 and KCM1003 respectively, and the site information is as follows:

TABLE 1SNP site information of broccoli variety' Zhe Qing 100

4) The primer sequence for detecting the SNP locus based on the KASP technology determined by the invention and the PIC, MAF and heterozygosity rate information after 392 parts of materials are genotyped 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 the KASP technical system, the purity of the ' Zheqing 100' 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 adaptor added to the upstream primers F1-3 is 5 'GAAGGTGACCAAGTTCATGCT-3'; the sequence of the universal adaptor added to the upstream primers H1-3 is 5.

5) The genotype data of the broccoli 'Zhe Qing 100' and its parents on the 3 pairs of SNP primers are as follows:

TABLE 3 genotype of Zhe Qing 100' and its parents

Example 2 identification of purities of 'Zhe Qing 100' broccoli hybrids

The embodiment specifically provides a method for identifying the purity of 'Zheqing 100' broccoli hybrid, which comprises the following steps:

extraction of DNA

Randomly selecting 180 seeds of the 'Zheqing 100' 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) 500ul of 2% CTAB was added and ground thoroughly with a mill.

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) Add 200. Mu.l of TE (pH 8.0) or ddH ₂ And O, fully dissolving for later use.

2. Genotyping assays Using the KASP technique

The purity of 'Zheqing 100' is identified by using a specific KASP marker consisting of markers KCM1001, KCM1002 and KCM 1003.

The method comprises the following specific steps:

1) Extracting the genomic DNA of the broccoli to be detected;

2) Adding a specific KASP Primer mix and a general KASP Master mix into the DNA template extracted in the step 1) for PCR amplification;

The KASP Master mix comprises the following components: universal FRET cassette fluorescent primer, ROX internal reference dye, klear Taq 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 PCR were: pre-denaturation at 94 ℃ for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 60s, and reducing the annealing temperature of each cycle by 0.6 ℃ for 10 cycles; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 60s, and 26 cycles in total.

3. Analysis of results

Through detection, 180 hybrid seeds 'Zheqing 100' to be detected and SNP marker genotyping results of parents thereof with the numbers of KCM1001, KCM1002 and KCM1003 are obtained.

TABLE 4 KASP genotyping results for 180 samples tested

Sample name	KCM1001	KCM1002	KCM1003
				Sample1	TC	GA	AG
Sample2	TC	GA	AG
				Sample3	TC	GA	AG
……	……	……	……
				……	……	……	……
Sample179	TC	GA	AG
				Sample180	TC	GA	AG
NTC	DropOut	DropOut	DropOut
				P1	TT	GG	AA
P2	CC	AA	GG

The statistical results show that: the genotyping results of 180 samples to be tested using 3 markers are TC, GA and AG, respectively, the male parents are TT, GG and AA, the female parents are CC, AA and GG, respectively, and the purity of the hybrid is 100% (fig. 1-3).

EXAMPLE 3 identification of Zhejiang blue 100

The broccoli seed to be tested and 48 seeds of the 'Zhe Qing 100' standard sample are randomly selected, DNA is prepared according to the method in the embodiment 2, and 3 SNP markers related to the invention are utilized to carry out 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 100' on 3 SNP sites

Number of	Seed to be tested	Standard sample
			KCM1001	TC	TC
KCM1002	AA	GA
			KCM1003	GG	AG

And (4) analyzing results: through the detection, the typing results of the broccoli seeds to be detected and the Zheqing 100' at 3 SNP sites are obtained, and the comparison analysis shows that: the difference between the 2 SNP sites of the hybrid to be detected and the 'Zhe Qing 100' indicates that the hybrid to be detected is not the real hybrid of the 'Zhe Qing 100'.

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.

Sequence listing

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Claims

1. The primer combination for identifying the broccoli variety Zhejiang blue 100 is characterized in that the primer combination is as follows:

the primer consists of forward primers F1 and H1 and a reverse primer C1, and the nucleotide sequence of the primer is shown as SEQ ID No. 1-3; the molecular marker of the primers F1, H1 and C1 is KCM1001, and the molecular marker KCM1001 has C/T polymorphism;

the primer consists of forward primers F2 and H2 and a reverse primer C2, and the nucleotide sequence of the primer is shown as SEQ ID No. 4-6; the molecular marker of the primers F2, H2 and C2 is KCM1002, and the molecular marker KCM1002 has A/G polymorphism;

the primer consists of forward primers F3 and H3 and a reverse primer C3, and the nucleotide sequence of the primer is shown as SEQ ID NO. 7-9; the molecular marker KCM1003 amplified by the primers F3, H3 and C3 is provided with A/G 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 100' is characterized by comprising a primer combination shown in SEQ ID No. 1-9.

4. The kit according to claim 3, wherein the 5 'ends of the primers F1, F2 and F3 are connected with a universal linker with the sequence shown in SEQ ID No.10, and the 5' ends of the primers H1, H2 and H3 are connected with a universal linker with the sequence shown in SEQ ID No. 11.

5. A method for identifying a broccoli variety 'Zheqing 100' is characterized by comprising the following steps:

1) Extracting the genomic DNA of the broccoli to be detected;

2) Performing PCR amplification using the primer combination of claim 2;

3) Analyzing the PCR amplification product by using a fluorescence detector; when the genotype results of KCM1001, KCM1002 and KCM1003 are TT, GG and AA respectively, determining that the broccoli to be detected is the 'Zhe Qing 100' male parent; when the genotype results of KCM1001, KCM1002 and KCM1003 are CC, AA and GG respectively, determining that the broccoli to be detected is the 'Zhe Qing 100' female parent; when the genotype results of KCM1001, KCM1002 and KCM1003 are TC, GA and AG respectively, the broccoli to be detected can be judged to be the 'Zhe Qing 100' hybrid F1; if the other conditions except the deletion genotype are not the same, the broccoli to be detected can be judged to be the heterotypic strain.

6. The method of claim 5, wherein the PCR reaction system is: 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.

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, annealing at 55 ℃ for 60s, and 26 cycles in total.