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A method for identifying the authenticity of lettuce hybrids and the combination of kasp primers used therefor

A primer combination and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as difficult data sharing, poor representativeness of results, and few detection sites, so as to save manpower , Simple operation, low cost effect

Active Publication Date: 2021-11-23
BEIJING AGRO BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There have been a lot of reports on the construction of variety fingerprints, purity identification, and genetic diversity analysis based on SSR markers. Difficult to achieve data sharing and other insurmountable defects

Method used

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  • A method for identifying the authenticity of lettuce hybrids and the combination of kasp primers used therefor
  • A method for identifying the authenticity of lettuce hybrids and the combination of kasp primers used therefor
  • A method for identifying the authenticity of lettuce hybrids and the combination of kasp primers used therefor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, for identifying the acquisition of the primer combination of lettuce

[0074] 1. Discovery of 18 SNP sites

[0075] The invention obtains 200 SNP sites with good polymorphism, uniform distribution on chromosomes and high specificity by performing a large number of sequence analysis and function analysis on the lettuce genome. Design 200 KSAP primer sets, and then conduct a large number of screenings to select 18 primer sets that have stable amplification effects, clear typing, and can distinguish the samples to be tested. For the basic information of the SNP sites corresponding to these 18 primer sets, see Table 1, wherein the position of the SNP site on the chromosome is determined with reference to the V8 version of the lettuce genome (URL: https: / / genomevolution.org / coge / GenomeInfo.pl?gid=28333).

[0076] Table 1. Basic information of 18 SNP sites

[0077] SNP site name Base type of SNP site Chromosome The location of the SNP locus on ...

Embodiment 2

[0089] Embodiment 2, lettuce DNA fingerprint database construction and application

[0090] 1. Construction of Lettuce DNA Fingerprint Library

[0091] 1. Genomic DNA of 41 kinds of lettuce seedling leaves shown in Table 3 were respectively extracted by CTAB method to obtain the corresponding genomic DNA of lettuce.

[0092] 2. Taking the genomic DNA of 41 kinds of lettuce as templates, 18 primer sets were used for PCR amplification (18 primer sets were in the same reaction system), and the genotypes at 18 SNP sites were detected. The fluorescence quantitative PCR instrument AB-Q6 Flex (Thermo Fisher Scientific (China) Co., Ltd.) or the ArrayTape platform (Douglas Scientific Company) was used.

[0093] The reaction system when using fluorescent quantitative PCR instrument AB-Q6 Flex to detect is 5 μ L, including the genomic DNA of 1.0 μ L lettuce (concentration is 50ng / μ L), 0.07 μ L primer mixture (the concentration of each primer in the primer mixture is 100pmol / L), 2.5 μ...

Embodiment 3

[0104] The application of the combination of primers obtained in embodiment 3 and embodiment 1 for identifying lettuce in identifying the authenticity of lettuce hybrids

[0105] The hybrid combinations of 32 lettuce hybrids are shown in Table 4, and all lettuce hybrids are hybrid F1.

[0106] Table 4

[0107]

[0108]

[0109] 1. Genomic DNA of 32 leaves of lettuce hybrids shown in Table 4 were respectively extracted by CTAB method to obtain the corresponding genomic DNAs of lettuce hybrids.

[0110] 2. Using genomic DNA of 32 lettuce hybrids as templates, 18 primer sets were used for PCR amplification (18 primer sets were in the same reaction system), and the genotypes at 18 SNP sites were detected. For the detection method, refer to Step 1 and 2.

[0111] 3. Obtain the genotypes of 32 lettuce hybrids at 18 SNP sites, that is, the DNA fingerprints of the 32 lettuce hybrids.

[0112] 4. Using the CTAB method to extract the genomic DNA of the lettuce seedling leaves t...

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Abstract

The invention discloses a method for identifying the authenticity of lettuce hybrids and the used KASP primer combination. The method comprises the steps of: respectively detecting the genotypes of the lettuce to be tested and 32 lettuce hybrids based on the 18 SNP sites, and then making the following judgments: if the lettuce to be tested is based on the genotypes of the 18 SNP sites and the 32 If a certain variety in the lettuce hybrid is exactly the same, then the lettuce to be tested and the lettuce variety belong to the same variety. The invention makes the operation of distinguishing the authenticity of the lettuce hybrid become accurate and convenient. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simple operation, saving manpower and material resources, and is not easily affected by the environment, and has very broad application prospects. The invention has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying the authenticity of lettuce hybrids and a KASP primer combination used therefor. Background technique [0002] Lettuce (Lactuca sativa L.) originated from the Mediterranean coast of Europe and West Asia, and was domesticated from wild species. Leaf lettuce (lettuce), a cultivated variety of lettuce, is one of the most widely planted vegetables worldwide and one of the most produced vegetables. The lettuce flower is tongue-shaped flower, the flower is small, and the stigma is wrapped in the anther. One flower is composed of more than 20 tongue-shaped florets. The flowers of lettuce cannot be castrated at the bud stage, but can only be castrated at the flowering stage, but the flowering time is very short, which brings inconvenience to the acquisition of true hybrids. At present, domestic lettuce is mainly conventional species. Driven by profit, c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 杨效曾李波杨飞申飞邓杨
Owner BEIJING AGRO BIOTECH RES CENT
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