Sample mixing detection method for detecting purity of corn seeds based on mSNP technology

A detection method, corn technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem that the change cannot be directly identified and analyzed, so as to reduce the use of primer pairs, reduce the Cost, effect of fine-grained detection of genetic variants

Pending Publication Date: 2021-12-24
石家庄博瑞迪生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this marker can effectively identify varieties whose phenotypes are difficult to identify, it cannot be directly identified and analyzed for changes in the DNA sequence or a chromosome that only involve a certain site in the genome

Method used

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  • Sample mixing detection method for detecting purity of corn seeds based on mSNP technology
  • Sample mixing detection method for detecting purity of corn seeds based on mSNP technology
  • Sample mixing detection method for detecting purity of corn seeds based on mSNP technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1: Obtaining method of specific primer

[0087] The method for obtaining specific primers of the present invention is specifically:

[0088] Using the whole genome resequencing data of maize, use BWA-mem (http: / / bio-bwa.sourceforge.net / ) to backpost to the maize reference genome, and use GATK (https: / / software.broadinstitute.org / gatk / ) for single nucleotide variation identification.

[0089] The set of identified single nucleotide variation sites, screening the minimum allelic variation frequency > 0.01, heterozygosity rate < 15%, deletion rate < 20%, combined single nucleotide variation sites, screening single nucleotide The number of sites is located in the segment of 3-15, that is, the mSNP (polynucleotide polymorphism) site. Compared with the traditional SNP (single nucleotide polymorphism) site, the mSNP site can maximize the Using the information obtained by each primer pair, that is, as many SNP sites as possible can be detected without changing the...

Embodiment 2

[0092] Embodiment 2: the primer set used for the detection of corn seed purity

[0093] The primer set used for the detection of corn seed purity includes primer pair 1F / R~20F / R, and in primer pair 1F / R~20F / R, not only includes the specific primer sequence shown in SEQIDNo.1~40, but also includes general Primer sequence, the F-terminal universal primer sequence of the F primer in the primer pair 1F / R~20F / R is shown in SEQIDNo.41; The sequence of the R-terminal universal primer of the R primer in the primer pair 1F / R~20F / R is as follows Shown in SEQIDNo.42;

Embodiment 3

[0094] Embodiment 3: the obtaining method of primer mixture:

[0095] After obtaining specific primers, design specific tag sequences, and then re-synthesize primers. At this time, specific tag sequences will be added during primer synthesis. In this example, 96 sets of specific tag combinations are used, specifically:

[0096] According to the combination of specific tags, each specific primer was synthesized into 10 primers with different target tags. The sequence form of the primers with the target tags is shown in Table 2. And all R (reverse primers), take 10 μl of each primer, and dilute to 10ml; the concentration of each primer is 0.1 μM, and this embodiment prepares a total of 96 sets of primer pairs containing specific label combinations, that is, the primer mixture;

[0097] Table 2 Primer Set

[0098]

[0099]

[0100] "FFF" is the F-terminal universal primer sequence, and the F-terminal universal primer sequence is AACGACATGGCTACGATCCGACTT, as shown in SEQ ID N...

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Abstract

The invention relates to a sample mixing detection method for detecting the purity of corn seeds based on an mSNP technology, which is carried out by utilizing primer pairs 1F/R-20F/R; the gene sequences of the primer pairs 1F/R-20F/R are shown as SEQ ID No.1-40; according to the invention, the mSNP technology is adopted, so that more SNP variations can be detected under the condition that amplicons are not changed; a sample mixing method is adopted for detection; the sequencing cost is reduced while the amplification workload is reduced; the detection speed is also increased; and detection of 1440 seeds can be completed within one day at most. According to the method, the single nucleotide polymorphism is directly read by using a sequencing technology; and the purity result is directly interpreted through a program, so that the result is visual and reliable, and the influence of subjective factors during the seed development period and the result interpretation is avoided.

Description

technical field [0001] The invention belongs to the field of seed purity detection, and in particular relates to a mixed-sample detection method for detecting the purity of corn seeds based on mSNP technology. Background technique [0002] Maize is one of the three major food crops in the world and has a wide range of applications in industries such as food, feed and energy. According to data from China Report Network, the sown area of ​​corn in my country reached 41.28 million hectares in 2019 2 , with a total output of 260 million tons. Maize is also one of the most successful crops for utilizing heterosis, and high-purity maize hybrid seeds are the basis for fully exerting hybrid vigor. Due to the large planting area of ​​corn, the demand for seeds is also large, and the quality of seeds is particularly critical. Therefore, ensuring the stable development of corn production and promoting the steady increase of corn production will greatly promote my country's social st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6869C12N15/11
CPCC12Q1/6895C12Q1/6869C12Q2600/13C12Q2600/156C12Q2531/113C12Q2523/308
Inventor 李凝许彦芬高苗刘景艺张丛张萌郝军会刘田龚舒
Owner 石家庄博瑞迪生物技术有限公司
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