CN110724758A - Method for identifying purity of Jingnongke 728 corn hybrid based on SNP marker - Google Patents
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Abstract
The invention provides a method for identifying purity of 728-maize hybrids of Jing nong Keke based on SNP markers, which is characterized in that a high-throughput KASP technical platform is adopted based on 384 SNP sites, comprehensive evaluation of test effect, biological characteristics and polymorphic parameters is carried out by using more than 500 samples, 4 SNP markers with high quality, high stability, high polymorphism and high heterozygosity are finally determined, and 12 primers with 3 primers for amplifying each SNP marker are designed, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 1-12. The purity of the seeds of Jingnong Ke 728 can be identified by utilizing the 4 pairs of primer combinations, and self-bred seedlings, backcrossed seedlings and heterotypic plants can be identified.
Description
Technical Field
The invention belongs to the technical field of crop molecular biology, and particularly relates to a method for identifying purity of Jingnongke 728 corn hybrid based on SNP markers.
Background
The seeds are basic production data of agriculture, and the yield increasing potential of fine varieties is easily influenced by the purity of the seeds. The purity identification is one of indexes for judging whether the quality of the commercial seeds is qualified or not, is an important means for preventing counterfeit and shoddy seeds from flowing into the market, and is an important way for controlling the product quality of seed enterprises. The method for identifying the seed purity with high speed, high efficiency, low cost and high flux is established, thereby being very beneficial to improving the seed quality monitoring, promoting the standardized production and operation and ensuring the vital interests of the seed enterprises and farmers.
The traditional field identification method needs to observe the characteristic specificity of the variety in the development stage, and has the defects of long period, large workload, easy environmental influence and the like. With the development of molecular biology technology, molecular markers based on DNA polymorphisms are becoming powerful tools for crop seed identification. The SNP (single nucleotide polymorphism) marker is considered to be one of the most suitable markers for seed purity detection due to the characteristics of co-dominant, allelic gene variation, easy realization of high-throughput detection and the like.
The purity identification and variety differentiation of the corn are key links in a corn seed quality control system. The corn hybrid seed Jingnongke 728 (Jing MC01, Jing 2416) is a good variety bred by the corn research center of agriculture and forestry academy of sciences of Beijing city, and passes through national approval (national approval of Jade 2012003) in 2012; has the outstanding advantages of early maturity, high quality, drought resistance, water conservation, density resistance, lodging resistance, high and stable yield, suitability for mechanically harvesting seeds, full-process mechanization and the like. Recommended as a corn leading variety by the agricultural rural department, listed as an updated new variety and a high-yield created leading variety by Beijing City, and listed as a Jingjin Ji integrated main-pushed corn new variety.
The method for identifying the purity of the corn seeds is reported to be field plot planting identification, isozyme protein electrophoresis and SSR marker identification, and the purity identification method based on the SNP marker is not reported. The reported method has the problems of long identification period, large workload, easy influence by environment or development stage, difficult formation of a high-throughput detection method and the like.
Disclosure of Invention
The invention aims to provide a method for identifying the purity of Jingnong 728 corn hybrids based on SNP markers, which is suitable for KASP technology, has the advantages of stability and accurate result, and can be used for identifying the purity of the Jingnong 728 corn hybrids and the purity of the Jingnong 728 corn hybrids in a high-throughput manner.
In order to realize the purpose of the invention, 384 SNP loci disclosed in the patent of 'maize DNA fingerprint library and variety molecule identification SNP core locus combination-maizeSNP 384' (ZL 201410756086.0) are used as an initial locus set, and 335 maize national examination hybrid SNP-DNA fingerprint data are established. 192 different corn hybrids are selected, DNA is extracted by adopting a rapid method, CTAB, a kit and various methods, based on a KASP technical system, the DNA extracted by different methods is used for verifying the typing effect, repeatability and stability of the loci, and a candidate locus set is obtained. Respectively evaluating polymorphism parameters PIC (polymorphic information index), MAF (low allele frequency) and DP (variety identification capability) of the primers by using 200 parts of inbred lines of different types of corns and 335 data of corn hybrids; further screening was based on PIC and MAF values greater than 0.35 and DP values greater than 0.5. And analyzing the site heterozygosis rate by using 335 parts of fingerprint data of the maize hybrid, wherein the heterozygosis rate value is more than 0.45 for screening.
Through the multi-stage evaluation screening, 4 pairs of primer combinations suitable for identifying the purity of the seeds of Jingnong 728 are determined based on the fingerprint data of the Jingnong 728 and the parents of the Jingnong 728; the Jingnongke 728 fingerprints of the 3 pairs of primers are heterozygous genotypes, and the parents are complementary homozygous genotypes (mainly used for identifying female parent selfing seedlings and backcrossing seedlings); 1 pair of primers of Jingnong family 728 and parents are the same homozygous genotype (mainly used for identifying heterotypic strains); the above 4 pairs of primers were used in combination to verify genotype data and identification results from each other.
Based on the research of the invention, the invention provides a corn SNP molecular marker combination for identifying the purity of 728 corn hybrids of Jingnongke, which is characterized by comprising 4 SNP molecular markers, wherein the information of the 4 SNP molecular markers is shown in the following table 1:
TABLE 1
Further, the invention finds that the determined 4 SNP markers have high quality, high stability, high polymorphism and high heterozygosity, and the main information is shown in Table 2:
TABLE 2
The 4 SNP molecular markers SNPCP _1, SNPCP _2, SNPCP _3 and SNPCP _4 are obtained by sequentially amplifying 4 specific primer groups, wherein each specific primer group comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences of the primers contained in the 4 specific primer groups are respectively shown as SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
Further, the invention provides a specific primer combination for identifying purity of Jingnongke 728 corn hybrid, which comprises 4 specific primer groups, wherein each group of the 4 specific primer groups comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences of the 4 specific primer groups containing primers are respectively shown as SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
Based on KASP technical system, purity of 728 corn hybrid of Jingnongke is identified, in the embodiment of the invention, 2 upstream primers are adopted, and universal joint sequences are respectively added at 5' ends of the upstream primers. F1 has an added universal linker sequence of 5'-GAAGGTGACCAAGTTCATGCT-3', and F2 has an added universal linker sequence of 5'-GAAGGTCGGAGTCAACGGATT-3'.
The invention also provides a specific primer combination for identifying the Jingnongke 728 maize inbred seedlings and backcross seedlings, which comprises 3 specific primer groups, wherein each group of the 3 specific primer groups comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences of the primers contained in the 3 specific primer groups are respectively shown as SEQ ID NO.1-3, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
The invention provides a specific primer combination for identifying 728 seed heteromorphic strains of Jingnong Keke, which comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences are respectively shown in SEQ ID NO. 4-6.
The invention provides application of the corn SNP molecular marker or the specific primer combination in identifying the purity of the corn seed or the purity of the hybrid seed 728 in the Jingnong family.
The invention provides application of the corn SNP molecular marker or the specific primer combination in identifying the Jingnong 728 corn inbred seedling, backcross seedling and/or heterotypic strain.
The invention provides application of the corn SNP molecular marker or the specific primer combination in the identification of 728 corns in Jingnong family or in the auxiliary breeding of the corn molecular marker.
Based on the research of the invention, the invention also provides a method for identifying the purity of the Jingnongke 728 corn hybrid seeds based on SNP markers, which comprises the steps of adopting a specific primer combination containing the 4 specific primer groups, taking the DNA of a corn seed sample to be detected as a template, carrying out PCR amplification based on KASP technology, judging whether the single plant is a normal plant, an inbred seedling or an abnormal plant according to the genotype data of the corn seed sample to be detected on each pair of primers, wherein the judgment standard is as follows:
and determining the number of the selfed seedling seeds and the number of the hybrid seeds according to the judgment result by combining the genotype data of the parents, and comprehensively judging the purity value of the seeds.
The experimental typing effect of 4 pairs of SNP primers on the KASP technical platform suitable for identifying the purity of the seeds of the Jingnong's 728 is shown in figures 1 to 4.
Purity calculation formula: p (%) [ (NT-NP-ND)/NT ] × 100%; wherein, P is the seed purity; NT number of seeds for detection; the number of NP self-bred seedlings; number of ND hybrid plants (including backcross seedlings and allotypic plants).
The 4 pairs of primers were analyzed in Jingnong 728, and if all individuals had genotypes of the SNPCP _1, SNPCP _2, SNPCP _3, SNPCP _4 primers AG, GG, CT, all individuals were normal Jingnong 728 individuals. If the genotype data of some single plants in the primers SNPCP _1, SNPCP _3 and SNPCP _4 is homozygous female parent Beijing MC01 genotype, namely GG, CC and CC, the detected single plants are self-bred seedlings. If some single plants are found in the genotype normal Jing agriculture 728 heterozygous genotype and the parent Jing MC01 homozygous genotype of the SNPCP _1, SNPCP _3 and SNPCP _4 primers, the detected single plants are backcrossed seedlings. If the genotype of some single plants in the SNPCP _2 primer is heterozygous genotype or another homozygous genotype, the detected single plants are heterotypic plants.
The invention is based on 384 SNP sites, adopts a high-flux KASP technical platform, utilizes more than 500 samples to carry out comprehensive evaluation on test effect, biological characteristics and polymorphic parameters, finally determines 4 SNP markers with high quality, high stability, high polymorphism and high heterozygosity, and designs a total of 12 primers for amplifying 3 primers of each SNP marker. By utilizing the 12 primers, the invention provides a rapid and efficient method for identifying the purity of the Jingnongke 728 corn hybrid based on SNP markers. The method can be used for identifying the seed purity of Jingnong 728, can identify self-bred seedlings, backcrossed seedlings and heterotypic plants, expands the purity identification mark types and methods of corn hybrid seeds by applying the method, and provides powerful technical support for popularization and planting of the Jingnong 728.
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FIG. 1 is a diagram of the typing effect of a specific primer group of SNPCP _1 locus for identifying the purity of seeds of Jingnong Ket 728 on a KASP technical platform. In the figure, the genotype of the upper left cluster is GG, the genotype of the middle cluster is AG, and the genotype of the lower right cluster is AA.
FIG. 2 is a diagram of the typing effect of the specific primer group of the SNPCP _2 locus on the KASP technical platform for identifying the purity of seeds of Jingnong Kege 728. In the figure, the genotype of the upper left cluster is TT, the genotype of the middle cluster is TG, and the genotype of the lower right cluster is GG.
FIG. 3 is a diagram of the typing effect of the specific primer group of the SNPCP _3 locus on the KASP technical platform for identifying the purity of seeds of Jingnong Kege 728. 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. 4 is a diagram of the typing effect of the specific primer group of the SNPCP _4 locus on the KASP technical platform for identifying the seed purity of the Jingnong family 728. 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.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples follow conventional experimental conditions. Those skilled in the art will appreciate that the details of the present invention not described in detail herein are well within the skill of those in the art.
If not otherwise stated, the biochemical reagents used in the examples of the present invention are commercially available, and the corn materials used are all commonly used corn known in the art.
Example 1 determination of SNP molecular markers and primers for identifying purity of Zea mays hybrid of Jingnongke 728
(1) Basal site: 384 SNP loci published by a 'maize DNA fingerprint library and variety molecule identification SNP core locus combination-maizeSNP 384' (disclosed in Chinese patent 201410756086.0) are used as an initial locus set, and 335 maize national censorship SNP-DNA fingerprint data are established.
(2) Testing and biological characterization of SNP sites: selecting 192 corn hybrids, extracting DNA by adopting a rapid method, CTAB, a kit and various methods, verifying the typing effect, the repeatability and the stability of the loci by using the DNA extracted by different methods based on a KASP technical system, and obtaining a candidate locus set.
(3) SNP site polymorphism evaluation: respectively evaluating polymorphism parameters PIC (polymorphic information index), MAF (low allele frequency) and DP (variety identification capability) of the primers by using 200 parts of inbred lines of different types of corns and 335 data of corn hybrids; further screening was based on PIC and MAF values greater than 0.35 and DP values greater than 0.5.
(4) Evaluation of SNP site heterozygosity rate and comprehensive distinguishing effect: and analyzing the site heterozygosis rate by using 335 parts of fingerprint data of the maize hybrid, wherein the heterozygosis rate value is more than 0.45 for screening.
(5) Determination of 4 pairs of SNP primers: through the multi-stage evaluation screening, 4 pairs of primer combinations suitable for identifying the purity of the seeds of Jingnong 728 are determined based on the fingerprint data of the Jingnong 728 and the parents of the Jingnong 728; the Jingnongke 728 fingerprints of the 3 pairs of primers are heterozygous genotypes, and the parents are complementary homozygous genotypes (mainly used for identifying female parent selfing seedlings and backcrossing seedlings); 1 pair of primers of Jingnong family 728 and parents are the same homozygous genotype (mainly used for identifying heterotypic strains); the above 4 pairs of primers were used in combination to verify genotype data and identification results from each other.
The information of 4 SNP loci suitable for purity identification of 728 seeds in Jingnong family is shown in Table 3. The genotype data of Jingnong 728 and its parents at 4 SNP sites are shown in Table 4. The experimental typing effect of 4 pairs of SNP primers on the KASP technical platform suitable for identifying the purity of the seeds of the Jingnong's 728 is shown in figures 1 to 4.
TABLE 3 four primer information for purity identification of seeds of Jingnong Ke 728
Note: the upstream primers are respectively added with universal joint sequences at the 5' ends. The added universal linker sequence of the upstream primer F1 is 5 'GAAGGTGACCAAGTTCATGCT 3', and the added universal linker sequence of the upstream primer F2 is 5 'GAAGGTCGGAGTCAACGGATT 3'.
TABLE 4 genotype data of Jingnongke 728 and its parents on 4 SNP primers
| Sample name | SNPCP_1 | SNPCP_2 | SNPCP_3 | SNPCP_4 |
| Jingnongke 728 | AG | GG | CT | CT |
| Jing MC01 | GG | GG | CC | CC |
| Jing 2416 | AA | GG | TT | TT |
Example 2 method for identifying purity of Jingnongke 728 corn hybrid by using SNP molecular marker provided by the invention
The scheme for identifying the seed purity of Jingnong Ke 728 by using the primer combination aiming at 4 SNP sites, which is designed in the embodiment 1, is as follows: synthesizing a primer according to the KASP technical requirement, wherein the primer is a common primer without a fluorescent group; purchasing a PCR amplification system MasterMix matched with the KASP technology; preparing a reaction system, and adding DNA, a primer and MasterMix; running an amplification reaction program; scanning the fluorescence signal in situ; carrying out data analysis to obtain genotype data; and (3) judging whether each single plant is a normal plant, a selfed seedling or an abnormal plant according to the genotype data of each single plant of Jingnongke 728 on each pair of primers, and finally comprehensively judging the purity value of the single plant. The specific operation is as follows:
(1) preparation of a sample to be tested:
randomly extracting at least 150 seeds from the sample to be detected, and finally obtaining samples of not less than 100 strains. Each individual plant may be subjected to DNA extraction using seeds, seedlings or leaf tissue. The specific steps of DNA extraction are carried out according to the identification standard of the corn DNA molecules (Wanfengge et al, 2014, corn variety identification technical specification SSR marking method, and agricultural industry standard of the people's republic of China), and the concentration of the working solution formed by diluting DNA is 20 ng/mu L.
(2) And (3) PCR amplification:
the PCR amplification system can be 1. mu.l, 3. mu.l or 10. mu.l, and the specific components are shown in Table 3 below. PCR amplification procedure: 15min at 94 ℃; 94 ℃ 20s, 61-55 ℃ 60s, 10 cycles (0.6 ℃ reduction per cycle); 94 ℃ for 20s, 55 ℃ for 60s, 26 cycles.
TABLE 5 PCR amplification System based on KASP technology System
| Microporous plate type | 1536 micro-porous plate | 384 microplates | 96 micro-porous plate |
| PCR system | 1μl | 3μl | 10μl |
| 2 XPCR premix | 0.5μl | 1.5μl | 5μl |
| Deionized water | 0.486μl | / | 3.36μl |
| Primer working solution | 0.014μl | 0.042μl | 0.14μl |
| DNA working solution | 1.5 μ l (drying) | 1.5μl | 1.5μl |
(3) Fluorescence in situ scan and data read:
the amplification product was scanned for fluorescence signal using a BMG Pheastar (LGC, Middlesex, UK) instrument to obtain raw data. The raw data is imported into Kraken software (LGC, UK) for analysis to obtain fingerprint data of each data point. And checking and revising the original typing result, and classifying the data points into AA, AB and BB types.
(4) Data analysis statistics and result judgment:
according to the detection results of the individual plants of the 4 groups of primers Jingnong 728 and the genotype data of the parents (see table 4), the purity of the seeds of the Jingnong 728 is comprehensively judged, and the original records need to distinguish normal plants, self-bred seedlings and other types of hybrid plants.
Purity calculation formula: p (%) [ (NT-NP-ND)/NT ] × 100%; wherein, P is the seed purity; NT number of seeds for detection; the number of NP self-bred seedlings; number of ND hybrid plants.
The 4 pairs of primers were analyzed in Jingnong 728, and if all individuals had genotypes of the SNPCP _1, SNPCP _2, SNPCP _3, SNPCP _4 primers AG, GG, CT, all individuals were normal Jingnong 728 individuals. If the genotype data of some single plants in the primers SNPCP _1, SNPCP _3 and SNPCP _4 is homozygous female parent Beijing MC01 genotype, namely GG, CC and CC, the detected single plants are self-bred seedlings. If some single plants are found in the genotype normal Jing agriculture 728 heterozygous genotype and the parent Jing MC01 homozygous genotype of the SNPCP _1, SNPCP _3 and SNPCP _4 primers, the detected single plants are backcrossed seedlings. If the genotype of some single plants in the SNPCP _2 primer is heterozygous genotype or another homozygous genotype, the detected single plants are heterotypic plants.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. The corn SNP molecular marker combination for identifying the purity of 728 corn hybrids of Jingnong Ket is characterized by comprising 4 SNP molecular markers, wherein the 4 SNP molecular markers have the following information:
2. the maize SNP molecular marker combination according to claim 1, wherein the 4 SNP molecular markers SNPCP _1, SNPCP _2, SNPCP _3 and SNPCP _4 are obtained by sequentially amplifying 4 specific primer sets, wherein the specific primer set comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences of the primers contained in the 4 specific primer groups are respectively shown in SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
3. A specific primer combination for identifying purity of 728-maize hybrid of Jingnongke comprises 4 specific primer groups, wherein each group of the 4 specific primer groups comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences of the 4 specific primer groups containing primers are respectively shown as SEQ ID NO.1-3, SEQ ID NO.4-6, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
4. A specific primer combination for identifying inbred seedlings and backcross seedlings of 728 corns in Jingnong Keke is characterized by comprising 3 specific primer groups, wherein each group of the 3 specific primer groups comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences of the primers contained in the 3 specific primer groups are respectively shown as SEQ ID NO.1-3, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
5. A specific primer combination for identifying 728 seed heteromorphic strains of the Jingnong Keke is characterized by comprising 2 upstream primers and 1 downstream primer; the nucleotide sequences are respectively shown in SEQ ID NO. 4-6.
6. Use of the maize SNP molecular marker of claim 1 or 2, or the specific primer combination of any one of claims 3-5, for identifying the seed purity or hybrid purity of zea mays 728.
7. Use of the maize SNP molecular marker of claim 1 or 2, or the specific primer combination of claim 3, to identify jingkong 728 maize inbred, backcrossed and/or allotypic strains.
8. Use of a maize SNP molecular marker according to claim 1 or 2, or of a specific primer combination according to any one of claims 3 to 5, in the identification of kyotocaceae 728 maize.
9. Use of the maize SNP molecular marker of claim 1 or 2, or the specific primer combination of any one of claims 3-5, for assisted breeding of maize molecular markers.
10. A method for identifying purity of Jingnongke 728 corn hybrid seeds based on SNP markers is characterized in that the specific primer combination of claim 3 is adopted, DNA of a corn seed sample to be detected is used as a template, PCR amplification is carried out based on KASP technology, according to genotype data of the corn seed sample to be detected on each pair of primers, whether a single plant is a normal plant, a self-bred seedling or an abnormal plant is judged, and the judgment standard is as follows:
and determining the number of the selfed seedling seeds and the number of the hybrid seeds according to the judgment result by combining the genotype data of the parents, and comprehensively judging the purity value of the seeds.
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| CN112080570A (en) * | 2020-10-26 | 2020-12-15 | 大连海洋大学 | KASP labeled primer combination for identifying hybrid stichopus japonicus in Zhongrussia and application thereof |
| CN113528620A (en) * | 2021-08-09 | 2021-10-22 | 上海市农业科学院 | Molecular marker primer group and kit for corn inbred line SHL03 variety protection and application thereof |
| CN113969322A (en) * | 2020-07-24 | 2022-01-25 | 北京市农林科学院 | SNP core locus and primer for identifying purity of corn hybrid and high-throughput purity identification method |
| CN117737296A (en) * | 2024-02-21 | 2024-03-22 | 北京康普森生物技术有限公司 | SNP marker for identifying purity of Qingzao 510 maize hybrid and application thereof |
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| CN113969322A (en) * | 2020-07-24 | 2022-01-25 | 北京市农林科学院 | SNP core locus and primer for identifying purity of corn hybrid and high-throughput purity identification method |
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| CN112080570A (en) * | 2020-10-26 | 2020-12-15 | 大连海洋大学 | KASP labeled primer combination for identifying hybrid stichopus japonicus in Zhongrussia and application thereof |
| CN113528620A (en) * | 2021-08-09 | 2021-10-22 | 上海市农业科学院 | Molecular marker primer group and kit for corn inbred line SHL03 variety protection and application thereof |
| CN117737296A (en) * | 2024-02-21 | 2024-03-22 | 北京康普森生物技术有限公司 | SNP marker for identifying purity of Qingzao 510 maize hybrid and application thereof |
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