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A SNP locus, primer set and application for identifying the purity of watermelon hybrids

A technology of hybrid purity and primer set, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of few applications and no simultaneous application, etc., achieve broad application prospects, and increase the speed of listing , the effect of simple operation

Active Publication Date: 2021-05-14
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, SNP markers are still rarely used in the industry of watermelon variety purity identification, and there is no SNP marker combination that is applicable to dozens or even hundreds of watermelon variety purity identification at the same time.

Method used

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  • A SNP locus, primer set and application for identifying the purity of watermelon hybrids
  • A SNP locus, primer set and application for identifying the purity of watermelon hybrids
  • A SNP locus, primer set and application for identifying the purity of watermelon hybrids

Examples

Experimental program
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Effect test

preparation example Construction

[0058] The preparation method of the above kit also belongs to the protection scope of the present invention, and the method includes packaging each primer in any one of the above primer sets separately.

[0059] In a fourth aspect, the present invention provides an authenticity detection method for identifying watermelon varieties, the method comprising the following steps:

[0060] S1, DNA extraction: extract and obtain the genomic DNA of N watermelon hybrids to be tested of a watermelon variety to be tested; N is a natural number greater than 95; the larger the value of N, the higher the accuracy of identifying the purity of the watermelon hybrids to be tested; If the value of N is too small, the purity detection is inaccurate.

[0061] S2. Target primer set screening:

[0062] S2-1: More than or equal to 8 strains, such as 8-12 strains (such as 8-10 strains, 10-12 strains, 8 strains, 10 strains or 12 strains, of the above-mentioned N strains of watermelon hybrids to be te...

Embodiment 1

[0083] Acquisition of SNP Sites and Primer Combinations Used to Identify the Purity of Watermelon Hybrids

[0084] 1. Determination of 8 SNP sites

[0085] Based on the resequencing data of 96 watermelons in our laboratory and the watermelon reference genome data, according to the screening conditions: MAF>0.1, deletion rate<0.1, heterozygosity<0.1, uniform distribution of chromosomes, and the Pearson correlation coefficient with the genetic distance of the whole gene SNP is greater than 0.9. The PCA clustering effect is good, the discrimination is high, and the 50bp sequences on both wings are conservative (no InDel, no SSR, no other SNPs), and a total of 32 high-quality SNPs were selected.

[0086] Use these 32 pairs of SNP-KASP primers to obtain the genotypes of 412 watermelon hybrid varieties, and then screen the optimal combination with good SNP typing effect and at least one heterozygous site in the 412 hybrid varieties, and finally determine that the present invention i...

Embodiment 2

[0099] This example is the validity test of the SNP primer combination developed in Example 1. The 412 tested watermelon hybrids were common good hybrids or hybrids introduced from abroad. The details are shown in Table 3:

[0100] Table 3: Basic information of 412 tested watermelon hybrids

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] 1. Obtaining the genomic DNA of the tested watermelon hybrids:

[0109] The genomes of 412 leaves of tested watermelon hybrids were extracted by cetyltrimethylammonium bromide (CTAB). DNA, the genomic DNA of the tested watermelon hybrids was obtained.

[0110] The operation of the CTAB method is as follows: quickly grind the mixed leaves into powder in liquid nitrogen, put them in a 1.5ml centrifuge tube; add 800 μl of CTAB buffer preheated to 65°C for extraction, and extract in a water bath at 65°C for 30 minutes Add an equal volume of chloroform-isoamyl alcohol mixed solution, wherein the volu...

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Abstract

The invention discloses a method for identifying the purity of watermelon hybrids and the SNP primer combination used therefor. The SNP primer combination provided by the present invention is composed of 8 primer sets; each primer set is composed of 3 primer sequences, which are used to amplify a SNP site; the nucleotide sequences of each primer are sequentially as shown in SEQ ID NO: 1 to Shown in SEQ ID NO:24. The SNP primer combination can be used for early identification at the seed or seedling stage of watermelon hybrids to ensure the purity of the hybrids, effectively protect the rights and interests of producers and breeders, and provide technical support for the quality management of watermelon varieties. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simple operation, saving manpower and material resources, etc., and has very broad application prospects.

Description

technical field [0001] The invention belongs to the field of molecular markers and detection thereof, in particular to a SNP site for identifying the purity of watermelon hybrids, a primer set, a kit, a detection method and an application. Background technique [0002] Watermelon (Citrullus lanatus) is native to Africa and belongs to Cucurbitaceae. my country is a big producer and consumer of watermelons. Since the popularization and popularization of first-generation watermelon hybrid varieties in the 1980s, the cultivation area of ​​watermelons has expanded rapidly, and the amount of seeds used and corresponding varieties have also increased. At present, there are more than 1,100 watermelon varieties that have passed identification, certification, and registration in the country, and the number of watermelon varieties sold on the market far exceeds this number. However, the quality of watermelon seeds on sale is uneven. One of the more prominent problems is that the purit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 温常龙张建杨静静张晓飞罗江李向晶
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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