mSNP technology based mixed sample detection method for detecting purity of zucchini seeds

A detection method, zucchini technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem that dominant markers cannot be distinguished from heterozygous and homozygous, and pedigree analysis provides insufficient information , Need isotopes and other issues, to achieve the effect of reducing the use of primer pairs, fine detection of genetic variation, and improving accuracy and sensitivity

Pending Publication Date: 2021-12-07
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 is a molecular marker technology invented in 1974. It has the advantages of good repeatability, co-dominance, and not affected by growth and development stages in seed identification; the disadvantage is that it does not provide sufficient information for pedigree analysis, and it is difficult to identify. It is difficult to operate in general detection rooms using isotopes, etc.
Compared with RFLP, RAPD has the advantages of requiring less DNA, simpler operation, and no need to use isotopes; the disadvantage is that dominant markers cannot distinguish heterozygous and homozygous types, and poor stability.
AFLP is a technology invented by Zabeau in the Netherlands in 1992. This technology can analyze crops with larger genomes, and its polymorphism is far more than RFLP and RAPD. The disadvantage is that it needs isotopes, etc.
SSR markers are co-dominant markers, rich in polymorphisms, and good in stability; however, primers must be designed for single-copy sequences at both ends of the microsatellites at each chromosomal locus, and the development of primers is difficult

Method used

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  • mSNP technology based mixed sample detection method for detecting purity of zucchini seeds
  • mSNP technology based mixed sample detection method for detecting purity of zucchini seeds
  • mSNP technology based mixed sample detection method for detecting purity of zucchini seeds

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Experimental program
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Effect test

Embodiment 1

[0088] Embodiment 1: Obtaining method of specific primer

[0089] The method for obtaining specific primers of the present invention is specifically:

[0090] Using the whole genome resequencing data of zucchini, use BWA-mem (http: / / bio-bwa.sourceforge.net / ) to post to the reference genome of zucchini, and use GATK (https: / / software.broadinstitute.org / gatk / ) for single nucleotide variation identification.

[0091] For the set of identified single nucleotide variation sites, screen the minimum allelic variation frequency > 0.02, heterozygosity rate < 15%, and deletion rate < 20%, combine the single nucleotide variation sites, and screen the single nucleotide The number of sites is located in the segment of 6-9, 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 wit...

Embodiment 2

[0094] Embodiment 2: Primer set used for the detection of zucchini seed purity

[0095] The primer set used for the purity detection of zucchini seeds includes the primer pair 1F / R~20F / R, and in the primer pair 1F / R~20F / R, it not only includes the specific primer sequence shown in SEQIDNo.1~40, but also includes the 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

[0096] Embodiment 3: the obtaining method of primer mixture:

[0097] 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:

[0098] 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;

[0099] Table 2 Primer Set

[0100] Primer pair F forward primer (5'~3') R reverse primer (5'~3') 1F / R FFFYYYCATGCTACTGGTGCTTCAAAGTATT RRRYYYGCATAGGGCAAAAATCCTAGCATAT 2F / R F...

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Abstract

The invention relates to an mSNP technology based mixed sample detection method for detecting the purity of zucchini seeds. The method is carried out by utilizing a primer pair 1F / R-20F / R, and the gene sequence of the primer pair 1F / R-20F / R is as shown in SEQ ID No.1-40. According to the invention, an mSNP technology is adopted, so that more SNP variations can be detected under the condition that amplicons are not changed; and a mixed sample 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 a 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 zucchini seeds based on mSNP technology. Background technique [0002] Zucchini (Cucubita pepo L.) is an annual plant of Cucurbitaceae squash, native to southern North America, so it is also called American squash. Zucchini has both nutritional value and health care function, and is loved by the public. It is planted in large areas all over our country and is one of the main varieties of vegetables in our country. Except for a small amount of imported varieties such as French winter jade and American jasper, other seeds on the market mainly rely on domestic cultivation, and the seed production areas are mainly concentrated in northwest my country and northern Shanxi Province. The two most important indicators of seed quality in seed production and trade are seed purity and authenticity. Using hybrid vig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6869C12N15/11
CPCC12Q1/6895C12Q1/6869C12Q2600/156C12Q2600/13C12Q2531/113C12Q2523/308C12Q2563/143C12Q2563/149C12Q2563/185
Inventor 许彦芬高苗刘景艺李凝张萌龚舒郝军会刘田张丛
Owner 石家庄博瑞迪生物技术有限公司
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