Molecular marker for corn variety identification as well as development method and application of molecular marker

Abstract

The invention relates to the technical field of biology, and provides a molecular marker for identifying maize varieties, which comprises 40 pairs of marker primers designed based on 40 SSR sites in the national standard of maize. The distribution of the molecular markers on the whole genome is completely consistent with the distribution of 40 national standard SSR sites of corn, and each chromosome corresponds to 4 pairs of markers on average. The invention also provides a development method and application of the molecular marker. According to the invention, through innovative means such as deep primer optimization and comprehensive sequence retrieval, the problem of dimer formation is ingeniously avoided, and 40 pairs of labeled primers are successfully developed and designed; based on the primers, by means of multiple PCR amplification, sequencing library construction, sequencing, biological information analysis and the like, the actual application requirements of corn variety authenticity identification, purity detection, germplasm resource identification and the like can be met; meanwhile, the method has the characteristics of low cost, simplicity in operation, high flux, high sensitivity and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a set of molecular markers for identifying corn varieties and a development method and application thereof. Background Art

[0002] In my country, corn is the largest crop, and accurate detection of its variety authenticity is crucial. For a long time, the authenticity detection of corn varieties has mainly relied on the SSR molecular marker method, and the detection standard followed is the "Technical Regulations for Corn Variety Identification SSR Marker Method" (NY / T 1432-2014). Looking back over the past 40 years, the detection method has continued to evolve, initially using polyacrylamide gel electrophoresis, then developing into capillary electrophoresis, and then transitioning to fluorescent labeling electrophoresis. However, these traditional detection methods have many shortcomings, the operation process is cumbersome and complicated, the detection cycle is lengthy, and the throughput is low, which makes it difficult to meet the growing detection needs.

[0003] With the innovation of technology, KASP (competitive amplification) detection method came into being. Its appearance significantly improved the detection throughput, and the operation was also simpler and easier, bringing new hope for corn variety detection. However, KASP detection is highly dependent on imported reagents, with high detection costs, and has encountered bottlenecks in large-scale promotion and application.

[0004] At the same time, high-throughput sequencing (NGS) technology has shown unique advantages in the field of SNP detection, with low cost, easy operation and extremely high throughput. However, when applied to SSR detection, it has exposed the problem of oversensitivity, which often requires additional correction of the test results, increasing the complexity and uncertainty of the result determination.

[0005] In addition, multiplex PCR is widely used in the capture amplification process. With its unique advantages, it greatly reduces the complexity of operation and has attracted much attention. However, multiplex PCR has strict requirements on primers. Once mixed primers produce dimers, it will seriously interfere with the amplification effect and affect the entire detection process. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a set of molecular markers for corn variety identification and their development method and application. Through innovative means such as in-depth primer optimization and comprehensive sequence retrieval, it cleverly avoids the problem of dimer formation and successfully develops and designs 40 pairs of marker primers; based on the above primers, and relying on multiple PCR amplification, sequencing library construction, sequencing and bioinformatics analysis, it can meet the actual application needs of corn variety authenticity identification, purity detection, germplasm resource identification, etc.; at the same time, the method has the characteristics of low cost, simple operation, high throughput and high sensitivity.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A set of molecular markers for corn variety identification includes 40 pairs of marker primers designed based on 40 SSR loci in the national corn standard, as shown in Table 1.

[0009] Table 1 Labeling primer information

[0010]

[0011]

[0012] As one of the preferred embodiments of the present invention, the distribution of the molecular markers on the whole genome is completely consistent with the distribution of 40 SSR loci in the national standard of corn, and each chromosome corresponds to 4 pairs of markers on average, and the specific marker position information is shown in Table 2. The size of the amplified product is between 129 and 417 bp, and the size of 80% of the amplified product is between 190 and 384 bp, which is suitable for the PE-150 sequencing platform and corresponding analysis.

[0013] Table 2 Marking position information

[0014]

[0015]

[0016] A molecular marker development method for corn variety identification comprises the following steps:

[0017] (1) Sequence information of 40 SSR loci in the national standard of maize was extracted to form a target locus data set; population variation data in the international maize genome database was used to form a variation data set; high-throughput sequencing data variation data analysis software GATK was used to perform variation analysis and screening, with the screening condition "minor allele frequency not less than 5%" to obtain candidate sequencing loci and variation information;

[0018] (2) Primer 3 software was used to design amplification primers for each target SNP site. The parameters set included: ① the length of the primer sequence was between 17 and 32 bp; ② the Tm value was between 60 and 64°C; ③ the product size was not less than 150 bp and not more than 500 bp; ④ the sequencing reads covered the target site;

[0019] (3) For each target site, three pairs of primers were designed, and then the ePCR software was used to detect the amplification specificity of each pair of primers. The possibility of dimer formation between primers was detected using a script, and finally the amplification primers for 40 SSR sites were obtained, namely the 40 pairs of marker primers shown in Table 1.

[0020] An application of the above-mentioned molecular markers for identifying corn varieties is characterized in that the genomic DNA of corn is amplified by mixed PCR using the 40 pairs of labeled primers, and the amplified products are sequenced and typed to obtain the polymorphism data of corn at the 40 SSR sites of the national standard, thereby forming the DNA fingerprint data of the corn; based on the DNA fingerprint data of the corn, subsequent analysis of the corn varieties can be carried out.

[0021] As one of the preferred embodiments of the present invention, the object of extracting the genomic DNA of corn is young and tender tissues of corn, more preferably dry corn seeds (endosperm), fresh leaves, young ears, seedlings or young and tender stem segments, old leaves, etc.

[0022] As one of the preferred embodiments of the present invention, the reaction system for PCR amplification is a 20uL system or a 10uL system; wherein the 20uL reaction system comprises: 2uL corn genomic DNA (50-100ng), 5uL 10mM dNTPs, 1uL 10mM forward primer, 1uL 10mM reverse primer, 2uL 10x amplification buffer, 0.5uL Taq DNA polymerase, 3uL 10mM MgCl 2 Finally, add ddH 2 D water to a final volume of 20uL; 10uL reaction system is reduced year-on-year.

[0023] As one of the preferred embodiments of the present invention, the reaction conditions of the PCR amplification are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 60 s, extension at 72°C for 20 s, for a total of 35 cycles; extension at 72°C for 5 min, and maintaining at room temperature.

[0024] Based on the above method, 100 to 20,000 corn DNA samples can be detected simultaneously.

[0025] As one of the preferred embodiments of the present invention, the subsequent analysis of the corn variety refers to one or more of corn variety authenticity identification, seed purity identification, molecular design breeding, backcross breeding and background selection.

[0026] As one of the preferred embodiments of the present invention, the method for identifying the authenticity of corn varieties is as follows: extracting the genomic DNA of the corn variety sample to be tested and the control sample respectively; using the above 40 pairs of labeled primers to amplify and sequence the genomic DNA of the corn sample to be tested and the control sample, thereby obtaining the genotype data of the above corn sample to be tested and the control sample at 40 loci for variety authenticity identification.

[0027] As one of the preferred modes of the present invention, the corn seed purity identification method is: according to the genotype data of the parents of the hybrid seeds of the variety to be detected at 40 loci, analyze and select SNP site primers that are polymorphic in the parents; use the selected polymorphic primers to perform PCR amplification sequencing analysis on the F1 hybrid seeds, analyze the relationship between the F1 seeds and the parents, and thus identify the purity of the corn variety seeds. In the present invention, the number of the selected polymorphic markers is preferably no less than 2 to 3.

[0028] As one of the preferred embodiments of the present invention, the method for molecular design breeding, backcross breeding and background selection of corn is as follows: according to the above method, 40-site genotype data of the corn variety to be bred are obtained to obtain the background genotype; according to the breeding goal, the target gene donor parent is selected, and the target gene donor parent is hybridized with the variety to be bred to establish a hybrid separation group or a backcross separation group; in the separation group, the prospect is screened by the target trait; in the selected separation group, the 40-site genotype data of the group individuals are obtained according to the above method; the individual genotype is compared with the obtained background genotype, and the individual with the least number of variable sites is selected for further breeding, until a stable individual containing the target trait gene and the background genotype is fully restored is obtained.

[0029] The advantages of the present invention compared to the prior art are:

[0030] (1) The present invention effectively avoids dimer formation by means of primer optimization, sequence retrieval, etc., and successfully develops and designs 40 pairs of marker primers covering the SSR loci in the "Technical Regulations for Identification of Maize Varieties SSR Marker Method"; using the marker primers, and through multiple PCR amplification, sequencing library construction, sequencing and bioinformatics analysis, etc., it can meet the actual application needs of maize variety authenticity identification, purity detection, germplasm resource identification, molecular breeding background selection, etc.;

[0031] (2) The molecular markers for corn variety identification provided by the present invention are based on high-throughput sequencing technology for marker detection, and through a single amplification reaction, 40 SSR loci can be detected simultaneously. The larger the sample size, the lower the cost. At the same time, it has the characteristics of simple operation, high throughput, high sensitivity, etc., and has extremely high promotion and application value.

[0032] (3) The present invention can realize the process and informationization of corn molecular design breeding, improve corn breeding efficiency, shorten the breeding cycle, and promote agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic diagram of the distribution of the 40 marker sites in Example 2 on the genome;

[0034] Figure 2It is the atlas information of some corn resources in Example 3 (in the figure, the same color of the same mark represents the same genotype, and different colors represent different genotypes);

[0035] Figure 3 This is the cluster analysis diagram of the corn samples in Example 3. DETAILED DESCRIPTION

[0036] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Example 1

[0038] A set of molecular markers for corn variety identification in this embodiment includes 40 pairs of marker primers designed based on 40 SSR loci in the national corn standard, as shown in Table 1.

[0039] The distribution of the molecular markers on the whole genome is completely consistent with the distribution of 40 SSR loci in the national standard of corn, and each chromosome corresponds to 4 pairs of markers on average. The specific marker position information is shown in Table 2. At the same time, combined with Table 2, it can be seen that the size of its amplified product is between 129 and 417 bp, and the size of 80% of the amplified products is between 190 and 384 bp, which is suitable for the PE-150 sequencing platform and corresponding analysis.

[0040] Table 1 Labeling primer information

[0041]

[0042]

[0043] Table 2 Marking position information

[0044] Primer name chromosome Physical Location Amplification product size GB.01 Chr.1 43914207_43914558 352bp GB.02 Chr.1 199519487_199519723 237bp GB.03 Chr.2 74090364_74090640 277bp GB.04 Chr.2 225870759_225871108 350bp GB.05 Chr.3 751790_752081 292bp GB.06 Chr.3 232377659_232377873 215bp GB.07 Chr.4 1284154_1284563 410bp GB.O8 Chr.4 172522807_172523190 384bp GB.09 Chr.5 28892818_28893138 321bp GB.10 Chr.5 213923412_213923673 262bp GB.11 Chr.6 2126021_2126205 185bp GB.12 Chr.6 151092363_151092627 265bp GB.13 Chr.7 1464003_1464226 224bp GB.14 Chr.7 173953563_173953729 167bp GB.15 Chr.8 165268302_165268538 237bp GB.16 Chr.8 178412077_178412292 216bp GB.17 Chr.9 68851643_68852059 417bp GB.18 Chr.9 121460654_121460928 275bp GB.19 Chr.10 5439596_5439816 221bp GB.20 Chr.10 134476747_134476936 190bp GB.21 Chr.1 228756898_228757063 166bp GB.22 Chr.1 279928282_279928493 212bp GB.23 Chr.2 2812983_2813242 260bp GB.24 Chr.2 210231389_210231619 231bp GB.25 Chr.2 230692420_230692583 164bp GB.26 Chr.3 205192852_205193085 234bp GB.27 Chr.4 32890631_32890902 272bp GB.28 Chr.4 224943201_224943390 190bp GB.29 Chr.5 12794900_12795178 279bp GB.30 Chr.5 67030592_67030720 129bp GB.31 Chr.6 44432358_44432620 263bp GB.32 Chr.6 166029502_166029727 226bp GB.33 Chr.7 10457665_10457882 218bp GB.34 Chr.7 160633569_160633738 170bp GB.35 Chr.8 13780331_13780522 192bp GB.36 Chr.8 179043680_179043881 202bp GB.37 Chr.9 4092504_4092703 200bp GB.38 Chr.9 135613487_135613762 276bp GB.39 Chr.10 2229791_2230100 310bp GB.40 Chr.10 115709777_115710111 335bp

[0045] Example 2

[0046] The development method of the above molecular markers in this embodiment is as follows:

[0047] 1. Label conversion and reference dataset acquisition

[0048] Based on the position and variation information of 40 pairs of SSR primers in the national standard for corn (existing technology), the genome of the published corn variety B73 was used as the "reference genome" (version: B73_v5.0, website: https: / / staging.maizegdb.org / genome / assembly / Zm-B73-REFERENCE-NAM-5.0), and the sequence information of 40 sites was extracted to form the target site data set.

[0049] The population variation data in the International Maize Genome Database (https: / / staging.maizegdb.org / ) was used to construct a variation data set. The high-throughput sequencing data variation data analysis software GATK (version 3.7) was used for variation analysis and screening (screening condition: the minor allele frequency was not less than 5%) to obtain candidate sequencing sites and variation information.

[0050] 2. Specific primer development:

[0051] Primer 3 software (version 2.5.0) was used to design amplification primers for each target SNP site. The parameters set included: ① the length of the primer sequence was between 17 and 32 bp; ② the Tm value was between 60 and 64°C; ③ the product size was not less than 150 bp and not more than 500 bp; ④ the sequencing reads must be able to cover the target site.

[0052] For each target site, three pairs of primers were designed, and then the amplification specificity of each pair of primers was tested using e-PCR software (version 2.3.12). The possibility of dimer formation between primers was tested using a script, and finally amplification primers for 40 sites were obtained. These 40 sites constituted a set of markers described in Example 1. The distribution of these 40 marker sites on the genome is shown in Figure 1 shown.

[0053] Example 3

[0054] A corn variety / resource identification in this embodiment.

[0055] Corn resources from different sources were selected as samples to be tested for variety / resource identification to verify the feasibility of the marker primers of the present invention. The specific operation was as follows:

[0056] 1. Extraction of genomic DNA from corn varieties / resources

[0057] The CTAB method was used to extract DNA from maize tissues.

[0058] 1. Select young corn tissue to extract genomic DNA.

[0059] 2. Reagent preparation:

[0060] CTAB buffer: 2% CTAB, 1.4 M NaCl, 100 mM Tris-HCl, 10 mM EDTA, pH 8.0;

[0061] Wash buffer: 75% ethanol;

[0062] TE buffer: 20 mM Tris-HCl, 1 mM EDTA, pH 8.0;

[0063] Precool anhydrous ethanol: Store anhydrous ethanol at -20℃ for 2h.

[0064] 3. Extract DNA from corn tissue:

[0065] Cut the young and tender tissues from corn into pieces and grind them in a liquid nitrogen environment; add preheated CTAB equivalent to the amount of tissue, and quickly place it in a 65℃ water bath for 30min to 1h, shaking every 5min; after centrifugation at 4℃ and 12000rpm for 10min, remove the supernatant and add an equal volume of chloroform and isoamyl alcohol (the volume ratio of chloroform and isoamyl alcohol is 24:1) mixed solution, and mix well; after centrifugation at 4℃ and 12000rpm for 15min, remove the supernatant and add twice the volume of ice-cold anhydrous ethanol, and place it at -20℃ for 1h; after centrifugation at 4℃ and 12000rpm for 10min, discard the supernatant, wash the precipitate with 75% ethanol and air-dry; add 5uL to 100uL of TE buffer to fully dissolve; detect the quality of DNA by agarose gel electrophoresis, and detect the concentration and purity by ultraviolet spectrophotometer; store the detected DNA at -20℃.

[0066] 2. Library Construction and Sequencing

[0067] The maize genomic DNA was mixed and PCR amplified using the 40 pairs of labeled primers described in Table 1 of Example 1. The PCR amplification reaction system (20uL) included: 2uL maize genomic DNA (50-100ng), 5uL 10mM dNTPs, 1uL 10mM forward primer, 1uL 10mM reverse primer, 2uL 10x amplification buffer, 0.5uL Taq DNA polymerase, 3uL 10mM MgCl 2 Finally, add ddH 2 O water to a final volume of 20uL. The reaction conditions were: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 55℃ annealing for 60s, 72℃ extension for 20s, a total of 35 cycles; 72℃ extension for 5min, maintained at room temperature.

[0068] After the PCR is completed, the product is recovered and qualified for quality inspection, and the paired-end (PE) sequencing library is constructed according to the Illumina library construction process; an equal amount of DNA from each sample is taken to construct a PE library, and PE150 sequencing is performed on the Illumina Hiseq sequencer.

[0069] 3. Target site genotype analysis

[0070] The raw data obtained by sequencing was quality controlled to obtain high-quality clean data, and then the clean data was decomposed into each target site using BWA software to obtain the SAM format comparison results, and then the SAM format file was converted into BAM format using samtools software, and then the reads in the BAM file were sorted using SortSam in the Picard tool to obtain the final BAM file. The genotype of each target site was determined using GATK, which constituted the DNA fingerprint of the maize germplasm.

[0071] Identification results:

[0072] Figure 2 This is part of the corn resource map information. Figure 3 This is the cluster analysis diagram of corn samples.

[0073] Combination Figure 2 and Figure 3 It can be seen that the 39 samples to be tested in this example are all corn resources from different sources, among which SCL23 and SCL35 are relatively closely related, and only 6 markers show differences.

[0074] Example 4

[0075] This embodiment provides a method for authenticating corn varieties.

[0076] Referring to the method in Example 3, the genomic DNA of the corn variety sample to be tested and the control sample were extracted respectively; and the genomic DNA of the corn sample to be tested and the control sample were amplified and sequenced using 40 pairs of labeled primers (Example 1), so as to obtain the genotype data of the corn sample to be tested and the control sample at 40 loci for comparison and variety authenticity identification.

[0077] Example 5

[0078] This embodiment provides a method for identifying the purity of corn seeds.

[0079] Referring to the method of Example 3, according to the genotype data of the parents of the hybrid seeds of the tested variety at 40 loci, the primers of the SNP loci that are polymorphic in the parents are analyzed and selected; the F1 hybrid seeds are subjected to PCR amplification and sequencing analysis using the selected polymorphic primers, and the relationship between the F1 seeds and the parents is analyzed, thereby identifying the purity of the corn variety seeds. In this embodiment, the number of polymorphic markers selected is not less than 2 to 3.

[0080] Example 6

[0081] This embodiment provides a method for molecular design breeding, backcross breeding and background selection of corn.

[0082] Obtain 40-site genotype data of the corn variety to be bred according to the method in Example 3 to obtain the background genotype; select the target gene donor parent according to the breeding goal, and hybridize it with the variety to be bred to establish a hybrid segregation population or a backcross segregation population; in the segregation population, perform prospect screening by target traits; in the selected segregation population, obtain 40-site genotype data of the individuals in the population according to the above method; compare the individual genotype with the obtained background genotype, and select the individual with the least number of variable sites for further breeding, until a stable individual containing the target trait gene and the background genotype is fully restored is obtained.

[0083] In summary, the present invention cleverly avoids the problem of dimer formation through innovative means such as in-depth primer optimization and comprehensive sequence retrieval, and successfully develops and designs 40 pairs of labeled primers; based on the above primers, and relying on multiple PCR amplification, sequencing library construction, sequencing and bioinformatics analysis, it can meet the actual application needs of corn variety authenticity identification, purity detection, germplasm resource identification, etc.; at the same time, the method has the characteristics of low cost, simple operation, high throughput and high sensitivity.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A set of molecular markers for corn variety identification, characterized in that: It includes 40 pairs of marker primers designed based on 40 SSR loci in the national standard of corn, as shown in Table 1. Table 1 Labeling primer information 2. The molecular marker for corn variety identification according to claim 1, characterized in that: The distribution of the molecular markers on the whole genome is completely consistent with the distribution of 40 SSR loci in the national standard of corn, and on average each chromosome corresponds to 4 pairs of markers. The specific marker position information is shown in Table 2. Table 2 Marking position information 3. A molecular marker development method for corn variety identification as claimed in claim 1 or 2, characterized in that: The steps include: (1) Sequence information of 40 SSR loci in the national standard of maize was extracted to form a target locus data set; population variation data in the international maize genome database was used to form a variation data set; high-throughput sequencing data variation data analysis software GATK was used to perform variation analysis and screening, with the screening condition "minor allele frequency not less than 5%" to obtain candidate sequencing loci and variation information; (2) Primer 3 software was used to design amplification primers for each target SNP site. The parameters set included: ① the length of the primer sequence was between 17 and 32 bp; ② the Tm value was between 60 and 64°C; ③ the product size was not less than 150 bp and not more than 500 bp; ④ the sequencing reads covered the target site; (3) For each target site, three pairs of primers were designed, and then the ePCR software was used to detect the amplification specificity of each pair of primers. The possibility of dimer formation between primers was detected using a script, and finally the amplification primers for 40 SSR sites were obtained, namely the 40 pairs of marker primers shown in Table 1.

4. An application of molecular markers for corn variety identification as described in 1 or 2, characterized in that: The 40 pairs of labeled primers are used to perform mixed PCR amplification on the genomic DNA of corn, and the amplified products are sequenced and typed to obtain the polymorphism data of corn at the 40 SSR sites of the national standard, forming the DNA fingerprint data of corn; based on the DNA fingerprint data of corn, subsequent analysis of corn varieties can be carried out.

5. The use of molecular markers for corn variety identification according to claim 4, characterized in that: The reaction system for PCR amplification is a 20uL system or a 10uL system; wherein, the 20uL reaction system includes: 2uL corn genomic DNA, 5uL10mM dNTPs, 1uL 10mM forward primer, 1uL 10mM reverse primer, 2uL 10x amplification buffer, 0.5uL Taq DNA polymerase, 3uL 10mM MgC12, and finally ddH2O water is added to the final volume of 20uL; the 10uL reaction system is reduced year-on-year.

6. The use of molecular markers for corn variety identification according to claim 4, characterized in that: The reaction conditions of the PCR amplification were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 60 s, and extension at 72°C for 20 s, for a total of 35 cycles; extension at 72°C for 5 min, and maintaining at room temperature.

7. The use of molecular markers for corn variety identification according to claim 4, characterized in that: The subsequent analysis work of the corn variety refers to one or more of the authenticity identification of the corn variety, seed purity identification, molecular design breeding, backcross breeding and background selection.

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