EST-SSR primer developing based on transcriptome sequence of wax gourd and application of EST-SSR primer

A technology of transcriptome sequence and EST-2, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve the problems that have not been reported in the application, and achieve strong polymorphism and high development cost , high versatility and conservative effect

Active Publication Date: 2020-06-19
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application of SSR primers developed based on wax gourd transcriptome sequence in the construction of wax gourd manual drawing variety identification diagram has not been reported

Method used

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  • EST-SSR primer developing based on transcriptome sequence of wax gourd and application of EST-SSR primer
  • EST-SSR primer developing based on transcriptome sequence of wax gourd and application of EST-SSR primer
  • EST-SSR primer developing based on transcriptome sequence of wax gourd and application of EST-SSR primer

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The present invention provides SSR primers developed based on the wax gourd transcriptome sequence and a method for constructing the artificially drawn variety identification diagram, including the following steps:

[0042] (1) Obtain the gene database of wax gourd samples;

[0043] (2) Use MISA software to search for SSR sites in the database described in step (1). Screening criteria: the minimum number of repetitions for single nucleotides is 10 times; the minimum number of repetitions for dinucleotides is 6 times; the minimum number of repetitions for three, four, five, and hexanucleotides is 5 times;

[0044] (3) Use Primer 3.0 to design SSR primers, and each SSR generates 3 sets of primers to generate candidate primers;

[0045](4) Randomly select 6 wax gourd resources, use CTAB method to extract wax gourd genomic DNA, use 1% agarose gel electrophoresis and spectrophotometer to detect the quality and concentration of DNA, use the method in step (3) The candidate ...

Embodiment 2

[0054] Example 2: Wax gourd EST-SSR amplification and polymorphism detection

[0055] According to the SSR sequence of wax gourd transcriptome, 40 wax gourd resources (Table 2) were amplified by PCR using EST-SSR primers (Table 1), and finally 3 pairs of primers with better specificity were screened out (Table 2). The results showed that the bands amplified by the three pairs of primers were clear and distinguishable. Further detection by a nucleic acid protein analyzer, the screened primers can detect polymorphic nucleic acid peaks. Figure 1-4 It is the detection result of primer 2. It can be seen that the lengths of polymorphic fragments detected by primer 2 in resource 1, resource 2, resource 3 and resource 10 are 216bp+304bp+333bp, 216bp+317bp+346bp, 218bp+322bp respectively +351bp and 213bp+300bp+329bp, the polymorphic nucleic acid peak map results are clear and clear, and the 4 resources can be distinguished. The length of the polymorphic fragment can be determined by...

Embodiment 3

[0064] Example 3: identification of wax gourd resources

[0065] According to the 3 pairs of SSR primers screened out, MCID method was used to identify wax gourd resources, and finally 40 wax gourd resources could be distinguished one by one ( Figure 5 ), and can basically reflect the previous relationship of most materials correctly, so the three pairs of primers can be used as EST-SSR primer combinations for wax gourd resources.

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Abstract

The invention discloses an EST-SSR primer developing based on transcriptome sequence of wax gourd and an application of the EST-SSR primer. The SSR locus of transcriptome sequence of the wax gourd isscreened, and primers are designed according to nucleotide sequences on the two sides of the SSR locus to obtain the EST-SSR primer. Three pairs of EST-SSR primer are used to amplify the wax gourd resources, and labeling results verified by an automatic nucleic acid protein analyzer can be applied to construction of artificial drawing of a schematic diagram of variety identification of the wax gourd. The wax gourd resources can be quickly and accurately distinguished according to the amplification results of the SSR, each resource can be effectively identified, and the EST-SSR primer can be used for germplasm identification, germplasm resources management, variety rights identification and protection and the like of varieties of benincasa plants in our country.

Description

technical field [0001] The invention belongs to the technical field of EST-SSR markers, in particular to EST-SSR primers developed based on wax gourd transcriptome sequences and applications thereof. Background technique [0002] Wax gourd belongs to the genus Benincasa Savi of Cucurbitaceae and is an annual vine herb. Winter melon is a vegetable variety traditionally grown in my country, with a cultivation history of more than 2,000 years. The breeding method of wax gourd mainly relies on conventional cross-breeding. Due to the high randomness and narrow genetic basis between varieties in the process of making new combinations in breeding, the heterosis is not obvious, which greatly affects the breeding efficiency. my country's wax gourd resources are relatively rich, and there are often phenomena of mutual introduction, cultivation, breeding and naming of different regions. In this process, the problem of homonym and synonym or homonym is prone to occur, which makes the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 叶新如王彬陈敏氡刘建汀曾美娟朱海生温庆放
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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