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

A detection method, technology of watermelon varieties, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve reaction conditions, extremely strict operation requirements, obstacles to the development and utilization of SSR technology, and development costs Advanced problems, to achieve the effect of reducing the number of primer pairs, fine detection of genetic variation, and low cost

Pending Publication Date: 2021-12-03
石家庄博瑞迪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RFLP polymorphisms are stable, but the amount of DNA required is large, the cost is high, and radioactive isotopes are required, which seriously restricts the application of this method
RAPD is easy to operate, has good polymorphism, and has a wide range of applications; however, it has extremely strict requirements on reaction conditions and operations, and poor repeatability
SSR has rich allelic variation, good reproducibility and co-dominant genetic characteristics; however, the development of SSR primers requires high cost, which has brought certain obstacles to the development and utilization of SSR technology

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0090] Embodiment 1: Obtaining method of specific primer

[0091] The method for obtaining specific primers of the present invention is specifically:

[0092] Using the whole genome resequencing data of watermelon, use BWA-mem (http: / / bio-bwa.sourceforge.net / ) to post to the watermelon reference genome, and use GATK (https: / / software.broadinstitute.org / gatk / ) for single nucleotide variation identification.

[0093] The set of identified single nucleotide variation sites, screening the minimum allelic variation frequency > 0.01, heterozygosity rate < 10%, deletion rate < 20%, combined single nucleotide variation sites, screening single nucleotide The number of sites is located in a section greater than 2, that is, the mSNP (polynucleotide polymorphism) site. Compared with the traditional SNP (single nucleotide polymorphism) site, the mSNP site can maximize the use of The information obtained by each primer pair is that as many SNP sites as possible can be detected under the ...

Embodiment 2

[0096] Embodiment 2: the primer set used for watermelon seed purity detection

[0097] The primer set used for watermelon seed purity detection includes primer pair 1F / R~22F / R, and in primer pair 1F / R~22F / R, not only includes the specific primer sequence shown in SEQIDNo.1~44, but also includes general Primer sequence, the F-terminal universal primer sequence of the F primer in the primer pair 1F / R~22F / R is shown in SEQIDNo.45; The sequence of the R-terminal universal primer of the R primer in the primer pair 1F / R~22F / R is as follows Shown in SEQIDNo.46;

Embodiment 3

[0098] Embodiment 3: the obtaining method of primer mixture:

[0099] 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:

[0100]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;

[0101] Table 2 Primer Set

[0102]

[0103]

[0104] "FFF" is the F-terminal universal primer sequence, and the F-terminal universal primer sequence is AACGACATGGCTACGATCCGACTT, as shown in SEQ ID ...

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Abstract

The invention relates to a sample mixing detection method for detecting the purity of watermelon seeds based on an mSNP technology, which is carried out by utilizing primer pairs 1F / R-22F / R, and the gene sequences of the primer pairs 1F / R-22F / R are shown as SEQ ID No.1-44. The mSNP technology is adopted, so that more SNP variations can be detected under the condition that amplicons are not changed; and the 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 watermelon seeds based on mSNP technology. Background technique [0002] Watermelon is an annual herb of Cucurbitaceae Cucurbitaceae. It has a long history of cultivation and is widely distributed. It is one of the top ten fruits in the world. The cultivated area and output of watermelon in my country rank first in the world. Since the first generation of watermelon hybrids was popularized in my country in the 1980s, the hybrids have made great contributions to the high yield, stable yield and high quality of watermelon, and have been widely used. Due to the many domestic seed management channels and the imperfect legal system of seed management, low-purity seeds are sometimes sold in the market. With the increase of planting area, the problem of the purity of hybrids has become more and more serious, c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2600/13C12Q2531/113C12Q2535/122C12Q2563/143C12Q2563/149C12Q2563/185
Inventor 郝军会许彦芬高苗李凝刘景艺张萌刘田龚舒张丛
Owner 石家庄博瑞迪生物技术有限公司
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