CN110872633B - Method for identifying purity of Jingke 968 corn hybrid based on SNP marker - Google Patents
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Abstract
The invention provides a method for identifying the purity of a Beijing 968 corn hybrid based on SNP markers, which is characterized in that a KASP (Kaasap technology platform) with high flux is adopted based on 384 SNP sites, more than 500 samples are utilized to comprehensively evaluate the test effect, the biological characteristics and the polymorphic parameters, 4 SNP markers with high quality, high stability, high polymorphism and high heterozygosity are finally determined, and 3 primers for amplifying each SNP marker are designed, wherein the total number of the primers is 12, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 1-12. The purity of the seeds of the Kyoco 968 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 the purity of a Kyoco 968 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 corn hybrid seed Jingke 968 (Jing 724, Jing 92) is a good variety bred by the corn research center of agriculture and forestry academy of sciences of Beijing, and passes through national approval (national approval of Yu 2011007) in 2011; has the advantages of high yield, high quality, multiple resistance, wide adaptability, easy seed production and the like. The planting area is popularized in the country to the first three digits, and the annual planting area reaches more than 3 million mu; the Jingke 968 is recommended by the agricultural Ministry of rural areas as a national corn leading variety for 5 consecutive years. Along with the continuous expansion of the planting area of the variety, the demand of seeds is correspondingly increased, and the research and development of a stable, reliable, rapid, simple and high-throughput method for identifying the purity of the seeds is beneficial to ensuring the popularization and planting of the variety.
The reported corn seed purity identification methods comprise field plot planting identification, isozyme protein electrophoresis and SSR marker identification methods, 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 the Jingke 968 seeds based on the SNP markers, which is suitable for the KASP technology, is stable and accurate in result, and can be used for identifying the purity of the Jingke 968 corn seeds 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 Beijing 968 seeds are determined based on the fingerprint data of the Beijing 968 and the parents thereof; the Beijing family 968 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 Jingke 968 and parents are all 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 an SNP molecular marker combination for identifying the purity of Jingke 968 corn hybrid, 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 as follows in the following 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 in 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 the purity of the Jingke 968 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, the purity of the Jingke 968 corn hybrid is identified, in the embodiment of the invention, 2 upstream primers F1 and F2 are adopted, and universal adaptor sequences are respectively added at the 5' ends of the upstream primers. 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'.
The invention also provides a specific primer combination for identifying the Beijing 968 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.4-6, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
The invention provides a specific primer combination for identifying a special-shaped strain of a 968 seed of a Beijing family, which comprises 2 upstream primers and 1 downstream primer; the nucleotide sequences are respectively shown in SEQ ID NO. 1-3.
The invention provides application of the corn SNP molecular marker or the specific primer combination in identifying the purity of the Jingke 968 corn seeds or the purity of hybrid seeds.
The invention provides application of the corn SNP molecular marker or the specific primer combination in identifying the Jingke 968 corn self-bred seedlings, backcrossed seedlings and/or heterotypic strains.
The invention provides application of the corn SNP molecular marker or the specific primer combination in Jingke 968 corn identification or corn molecular marker-assisted breeding.
Based on the research of the invention, the invention also provides a method for identifying the purity of the Jingke 968 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 Beijing family 968 seeds is shown in figures 1-4.
The purity calculation formula is as follows: 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 primer pairs were analyzed in Jingke 968, and if all individuals had genotypes with the SNPCP _1, SNPCP _2, SNPCP _3, SNPCP _4 primers GG, GT, CT, all individuals were normal Jingke 968 individuals. If the genotype data of some single plants in the primers SNPCP _2, SNPCP _3 and SNPCP _4 is homozygous female parent Beijing 724 genotype, namely GG, CC and CC, the detected single plants are self-bred seedlings. If some single plants are found in the genotype normal Jingke 968 heterozygous genotype and the parent Jing 724 homozygous genotype of the SNPCP _2, SNPCP _3 and SNPCP _4 primers, the detected single plants are backcrossed seedlings. If the genotype of some single plants in the SNPCP _1 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 Jingke 968 corn hybrid based on SNP markers. The method can be used for identifying the purity of the seeds of the Kyoco 968, and can identify self-bred seedlings, backcrossed seedlings and heterotypic plants, the application of the method expands the purity identification mark types and methods of the corn hybrid seeds, and powerful technical support is provided for the popularization and planting of the Kyoco 968.
Drawings
FIG. 1 is a diagram of the typing effect of the specific primer group of SNPCP _1 locus on KASP technical platform for identifying the purity of seeds of Kyoco 968. 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 showing the typing effect of the specific primer group of SNPCP _2 locus for identifying the purity of seeds of Kyoco 968 on the KASP technical platform. 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 showing the typing effect of the specific primer group of SNPCP _2 locus for identifying the purity of seeds of Kyoto 968 on the KASP technical platform. 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 SNPCP _4 site for identifying the purity of the seeds of Kyoco 968 on the KASP technical platform. 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 the purity of Kyoto 968 maize hybrids
(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: 192 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 typing effect, the repeatability and the stability of the sites are verified by using the DNA extracted by different methods, and a candidate site set is obtained.
(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 multistage evaluation screening, 4 pairs of primer combinations suitable for identifying the purity of the Kyoco 968 seeds are determined based on the fingerprint data of Kyoco 968 and the parents thereof; the Beijing family 968 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 Kyoco 968 and the same homozygous genotype of the parents (mainly used for identifying the heterotypic strain); 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 sites suitable for identifying the purity of the Kyoto 968 seeds is shown in the following table 3. Genotype data of kyoto 968 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 Beijing family 968 seeds is shown in figures 1-4.
TABLE 3 four primer information suitable for seed purity identification of Kyoto 968
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 Kyoco 968 and its parents on 4 SNP primers
| Sample name | SNPCP_1 | SNPCP_2 | SNPCP_3 | SNPCP_4 |
| Jingke 968 | GG | GT | CT | CT |
| Jing 724 | GG | GG | CC | CC |
| Jing 92 | GG | TT | TT | TT |
Example 2 method for identifying purity of Jingke 968 corn hybrid by using SNP molecular marker provided by the invention
The protocol for identifying the seed purity of the Jingke 968 by using the primer combination aiming at 4 SNP sites designed in the example 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 judging whether each single plant is a normal plant, a selfed seedling or an abnormal plant according to the genotype data of each pair of primers of each single plant of the Kyoco 968, 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:
as the PCR amplification system, 1. mu.l, 3. mu.l, and 10. mu.l systems were used, 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 KASP-based technical System, PCR amplification 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 4 groups of primers determined in the example 1 on each individual plant of the Jingke 968, and by combining the genotype data of the parents (see Table 4), the purity of the seeds of the Jingke 968 is comprehensively judged, and the original record needs 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 primer pairs were analyzed in Jingke 968, and if all individuals had genotypes with the SNPCP _1, SNPCP _2, SNPCP _3, SNPCP _4 primers GG, GT, CT, all individuals were normal Jingke 968 individuals. If the genotype data of some single plants in the primers SNPCP _2, SNPCP _3 and SNPCP _4 is homozygous female parent Beijing 724 genotype, namely GG, CC and CC, the detected single plants are self-bred seedlings. If some single plants are found in the genotype normal Jingke 968 heterozygous genotype and the parent Jing 724 homozygous genotype of the SNPCP _2, SNPCP _3 and SNPCP _4 primers, the detected single plants are backcrossed seedlings. If the genotype of some single plants in the SNPCP _1 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 (4)
1. The application of a specific primer combination in identifying the purity of a Jingke 968 corn hybrid, wherein the specific primer combination comprises 4 specific primer groups, and 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; the 4 specific primer groups respectively amplify 4 SNP molecular markers, and the 4 SNP molecular markers are respectively:
2. the application of a specific primer combination in identifying inbred seedlings and backcrossed seedlings of Kyoco 968 corn female parents, wherein the specific primer combination comprises 3 specific primer groups, and each specific primer 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.4-6, SEQ ID NO.7-9 and SEQ ID NO. 10-12.
3. The application of a specific primer combination in identifying Peking 968 maize heterotypic strains, wherein the specific primer combination contains 2 upstream primers and 1 downstream primer; the nucleotide sequences are respectively shown in SEQ ID NO. 1-3.
4. A method for identifying the purity of Jingke 968 corn hybrid seeds based on SNP markers is characterized in that the specific primer combination mentioned in the application of claim 1 is adopted, DNA of a Jingke 968 corn seed sample to be detected is used as a template, PCR amplification is carried out based on KASP technology, according to the genotype of the corn seed sample to be detected, whether a single plant developed by the corn seed sample to be detected is a normal plant, a female parent self-bred seedling, a backcrossed seedling or an abnormal plant is judged, and the judgment standard is as follows:
if the genotypes of four SNPs of a single plant to be detected, namely SNPCP _1, SNPCP _2, SNPCP _3 and SNPCP _4, are GG, GT, CT and CT respectively, the single plant is a normal Kyoco 968 single plant; if the genotypes of the single plant to be detected at three SNP sites of SNPCP _2, SNPCP _3 and SNPCP _4 are homozygous female parent Beijing 724 genotypes, namely GG, CC and CC, the detected single plant is a female parent selfing seedling; if the genotype of the single plant to be detected at one or two of the three SNP loci SNPCP _2, SNPCP _3 and SNPCP _4 is the normal Jingke 968 heterozygous genotype, and the genotypes of the other SNP loci are the female parent Jing 724 homozygous genotypes, the detected single plant is a backcross seedling; if the genotype of the single plant to be detected at the SNPCP _1 locus is a heterozygous genotype or another homozygous genotype except GG, the detected single plant is a heterotypic plant;
determining the seed number of the inbred seedling of the female parent and the seed number of the hybrid plant according to the judgment result and the genotype data of the parents, and comprehensively judging the purity of the seeds;
purity calculation formula: p (%) = [ (NT-NP-ND)/NT ]. times.100%; wherein, P is the seed purity; NT is the number of seeds to be detected; NP is the seed number of the female parent selfing seedling; ND is the number of the seeds of the hybrid plants, and the number of the seeds of the hybrid plants is the sum of the number of the seeds of the backcrossed seedlings and the number of the seeds of the heterotypic plants.
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| CN111850158B (en) * | 2020-07-08 | 2022-09-27 | 北京市农林科学院 | Specific SNP locus combination for purity identification of common wheat conventional variety and identification method |
| 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 |
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