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SNP loci and primer groups for identifying purity of tomato hybrid and application

A hybrid hybrid purity, primer set technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem of low detection accuracy, unstable detection results, and failure to find primer combinations question

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

However, at present, there is still no research on the purity identification of more varieties using molecular markers, and no primer combination that can identify more varieties has been found.
At the same time, the amplification results of SSR primers currently used have problems of false positives and false negatives, and the detection results are not stable enough; while InDel markers have strong stability, but there are relatively few sites, and the detection accuracy is not high.
However, there is no report on the use of SNP markers to identify the purity of tomato hybrids

Method used

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  • SNP loci and primer groups for identifying purity of tomato hybrid and application
  • SNP loci and primer groups for identifying purity of tomato hybrid and application
  • SNP loci and primer groups for identifying purity of tomato hybrid and application

Examples

Experimental program
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Effect test

preparation example Construction

[0055] 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.

[0056] In a fourth aspect, the present invention provides an authenticity detection method for identifying tomato varieties, the method comprising the following steps:

[0057] S1, DNA extraction: extract and obtain the genomic DNA of N strains of tomato 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 tomato hybrids to be tested; If the value of N is too small, the purity detection is inaccurate.

[0058] S2. Target primer set screening:

[0059] S2-1: 8 or more, for example, 8-12 strains (such as 8-10 strains, 10-12 strains, 8 strains, 10 strains or 12 strains, among the above-mentioned N strains of tomato hybrids to be tested, the number of samples The genomic DNA that is conveni...

Embodiment 1

[0080] Acquisition of SNP Sites and Primer Combinations Used to Identify the Purity of Tomato Hybrids

[0081] 1. Determination of 8 SNP sites

[0082] Based on 96 tomato resequencing data in the inventor's laboratory and tomato reference genome data, 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 genome 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.

[0083] Use these 32 pairs of SNP-KASP primers to obtain the genotypes of 163 tomato hybrid varieties, and then screen the optimal combination with good SNP typing effect and at least one heterozygous site in the 163 hybrids, and finally determine that the present invention is suitable for Eight SNP loci for purity identification of...

Embodiment 2

[0095] This example is the validity test of the SNP primer combination developed in Example 1. The 163 tested tomato hybrids were common good hybrids or hybrids introduced from abroad. The details are shown in Table 3:

[0096] Table 3: Basic information of 163 tested tomato hybrids

[0097]

[0098]

[0099]

[0100] 1. Obtaining the genomic DNA of the tested tomato hybrids:

[0101] The genomes of 163 leaves of the tested tomato hybrids were extracted by the cetyltrimethylammonium bromide (CTAB) method (30 seeds of each hybrid were taken to grow true leaves, and an equal amount of leaves were picked and mixed) DNA, the genomic DNA of the tested tomato hybrids was obtained.

[0102] 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 chl...

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Abstract

The invention discloses a method for identifying the purity of tomato hybrids and an SNP primer combination used by 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 can be used for early identification in the seed or seedling stage of tomato hybrids, the purity of the hybrids is guaranteed, the rights and interestsof producers and breeders are practically protected, and technical support is provided for seed quality management of tomato varieties. The method provided by the invention has the advantages of highefficiency, accuracy, low cost and the like, and has a very wide application prospect.

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 tomato hybrids, a primer set, a kit, a detection method and an application. Background technique [0002] Tomato (Solanum lycopersicum) belongs to Solanaceae Solanaceae annual or perennial herbaceous plants, native to Central and South America. Because of its unique taste, rich in trace elements needed by the human body, and high nutritional value, it is deeply loved by consumers. Tomato has only been cultivated for hundreds of years, but it has been widely distributed all over the world and is the second largest vegetable crop in the world. The annual output of tomatoes in my country exceeds 50 million tons, and a considerable part is planted and harvested by hybrid varieties. Tomato hybrids need to be castrated during the seed production process, but in actual production, the seed purity is often reduced due to im...

Claims

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Application Information

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Patent Type & Authority Applications(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杨明珠
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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