Method for identifying authenticity of watermelon varieties and SNP (Single Nucleotide Polymorphisms) primer combination special for method
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A primer combination, primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc. question
Active Publication Date: 2019-05-03
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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[0004] At present, the DNA molecular detection of watermelon variety identification in my country is mainly based on the SSR molecular marker method, but the research and development of SSR primers did not refer to the variation group information of the watermelon genome, and there are unreal variations; The sales varieties in the market; in addition, limited by the SSR detection method, it is easy to lead to false, false positive, and false negative results; it cannot adapt to the requirements of automation, high-throughput, and large-scale
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Embodiment 1
[0039] Embodiment 1, the acquisition of the primer combination that is used to identify the authenticity of watermelon varieties
[0040] 1. Discovery of 32 SNP sites
[0041] The present invention obtains 32 SNP sites based on the resequencing data of 17 watermelon representative resources. These 17 watermelon resources are rich in types, covering East Asian type (6), American type (5) and wild type (6), basically including the main ecological types of watermelon, in terms of growth period, fruit shape, peel thickness, flesh It has high genetic diversity in terms of agronomic traits such as color, seed size, and sugar content, and is representative of watermelon germplasm.
[0042] Specifically, the screening criteria for SNP loci are as follows: evenly selected positions throughout the genome, good polymorphism, low heterozygosity, MAF>0.3, good PCA clustering effect, high discrimination, and conservative 50bp sequences on both wings (no InDel , no SSR, no other SNP).
[...
Embodiment 2
[0055] The validity check of the primer combination that embodiment 2, embodiment 1 develop
[0056] The basic information of the 214 tested watermelon varieties in this example is shown in Table 3. The 214 tested watermelon varieties are all common fine varieties or some imported varieties from abroad.
[0057] Table 3. Basic information of 214 tested watermelon varieties
[0058]
[0059]
[0060]
[0061] 1. Obtaining the genomic DNA of the tested watermelon varieties
[0062] Genomic DNA of 214 leaves (true leaves of 30 seeds mixed) of 214 tested watermelon varieties were extracted by CTAB method to obtain genomic DNA of tested watermelon varieties.
[0063] The quality and concentration of the genomic DNA of the tested watermelon varieties must meet the requirements of PCR, and the standard of compliance is: agarose electrophoresis shows a single DNA band without obvious dispersion; the UV spectrophotometer Nanodrop2000 (Thermo) detects that the A260 / A280 ratio ...
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Abstract
The invention discloses a method for identifying authenticity of watermelon varieties and an SNP (Single Nucleotide Polymorphisms) primer combination special for the method. The SNP primer combinationprovided by the invention consists of 32 primer sets, each primer set consists of 3 primer sequences and is used for amplifying an SNP site, and the nucleotide sequences of the primers in the 32 primer sets are sequentially as shown in a sequence 1 to a sequence 96 in a sequence table. The SNP primer combination provided by the invention can be used for performing early identification on the watermelon varieties at the seed stage or seedling stage, so that the authenticity of the varieties can be guaranteed, the benefits of producers and breeders can be effectively protected, and a technicalsupport can be provided for protecting watermelon seed resources and new varieties. Through the adoption of the method provided by the invention, unknown watermelon varieties can be identified, and the authenticity of the known watermelon varieties can be identified. The method provided by the invention has the advantages of being high in flux, accurate, low in cost, and simple to operate, and saving human resources, and material resources and the like, and has great application prospects.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying the authenticity of watermelon varieties and a combination of special primers. Background technique [0002] my country is the largest country in watermelon production and consumption, with an annual planting area of 1.828 million hectares, accounting for 7.9% of all vegetable cultivation areas. From the 1950s to the present, my country's watermelon breeding has achieved at least 5 varieties of replacements, bred the main watermelon varieties in different periods, and greatly promoted the development of my country's watermelon industry. But problems also came one after another. Due to the incoordination of variety approval among various provinces and cities, the variety management and germplasm monitoring of watermelon failed to follow up in time, resulting in a large number of phenomena with different names and different names of the same speci...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 许勇温常龙杨静静张建张海英孙宏贺郭绍贵
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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