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SNP loci and primer groups for identifying purity of pepper hybrids and application

A hybrid hybrid purity, primer set technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., can solve the problems of limited distribution, limited amplification methods, and inconvenient use methods.

Active Publication Date: 2021-01-08
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the distribution of markers such as SSR and Indel is relatively limited, and limited by the amplification method, there are certain errors in it, and some methods of use are not easy enough

Method used

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  • SNP loci and primer groups for identifying purity of pepper hybrids and application
  • SNP loci and primer groups for identifying purity of pepper hybrids and application
  • SNP loci and primer groups for identifying purity of pepper hybrids and application

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] 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.

[0060] In a fourth aspect, the present invention provides an authenticity detection method for identifying watermelon germplasm resources, the method comprising the following steps:

[0061] S1, DNA extraction: extract and obtain the genomic DNA of N strains of pepper hybrids 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 pepper hybrids to be tested; If the value of N is too small, the purity detection is inaccurate.

[0062] S2. Target primer set screening:

[0063] 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 pepper hybrids to be tested, the number of samples The genomi...

Embodiment 1

[0083] Obtaining the SNP site and its primer combination for identifying the purity of pepper hybrids

[0084] 1. Determination of 8 SNP sites

[0085] Based on the resequencing data of 30 peppers in the inventor's laboratory and the reference genome data of peppers, according to the screening conditions: MAF>0.1, deletion rate<0.1, heterozygosity<0.1, uniform distribution of chromosomes, and Pearson correlation coefficient with the genetic distance of the whole gene SNP Greater than 0.9, good PCA clustering effect, high discrimination and conservative 50bp sequences on both wings (no InDel, no SSR, no other SNP), a total of 32 high-quality SNPs were selected. Use these 32 pairs of SNP-KASP primers to obtain the genotypes of 82 capsicum hybrid varieties, and then screen the optimal combination with good SNP typing effect and at least one heterozygous site in the 82 hybrids, and finally determine the present invention. Eight SNP loci suitable for purity identification of peppe...

Embodiment 2

[0097] This example is the validity test of the SNP primer combination developed in Example 1. The 82 pepper hybrids tested were common excellent hybrids or hybrids introduced from abroad. The details are shown in Table 3:

[0098] Table 3: Basic information of 82 pepper hybrids tested

[0099]

[0100]

[0101] 1. Obtaining the genomic DNA of the hybrid capsicum species for testing:

[0102] The genomes of 82 leaves of pepper hybrids tested were extracted by cetyltrimethylammonium bromide (CTAB). DNA, the genomic DNA of the tested pepper hybrids was obtained.

[0103] 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 volume ratio of chloroform and isoamyl alcohol is 24:1, after mixing...

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Abstract

The invention discloses a method for identifying the purity of pepper hybrids and an SNP primer combination used in the method. The SNP primer combination provided by the invention is composed of eight primer groups, wherein each primer group consists of three primer sequences and is used for amplifying one SNP locus; and the nucleotide sequences of the primers are sequentially shown as SEQ ID NO:1 to SEQ ID NO: 24. The SNP primer combination is confirmed to be suitable for purity identification of 82 pepper varieties, is stable and accurate in detection effect and high in detection speed, can be identified in seeds or seedling stage, is suitable for high-throughput detection equipment, is simple to operate, is time-saving and labor-saving, has a wide application prospect, and can providetechnical support for seed quality management of the pepper varieties.

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 pepper hybrids, a primer set, a kit, a detection method and an application. Background technique [0002] Pepper (Capsicum annuum) is one of the vegetable crops with the largest planting area in my country. In recent years, the planting area has exceeded 2 million hectares, and its output value has been in the forefront all year round. It has high nutritional value and wide application, and occupies an important share in both the fresh food market and the processing market. Since the implementation of the "Registration Measures for Non-Major Crop Varieties", 2,464 pepper varieties have been announced and registered nationwide, and the number of applications has exceeded 4,000. It is one of the vegetable crops with the largest number of applications and registrations. [0003] At present, most of the pepper varieties ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2563/107C12Q2535/101
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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