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
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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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