A kind of camphor ssr primer combination and its variety identification method
A technique for primer composition and species molecular identification, applied in biochemical equipment and methods, microbe measurement/inspection, recombinant DNA technology, etc., to achieve the effects of convenient sampling, convenient material collection, and important economic benefits
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Embodiment 1
[0030] Using the Illumina HiSeq 2500 sequencing platform, sequence the DNA of the leaves of Cinnamomum camphora to obtain the transcriptome sequence of Cinnamomum camphora, use MISA software to search for SSR sites, randomly select 100 SSR sites, and use Primer5.0 software to design primers , there are 74 pairs of primers that can amplify stably. Randomly select 6 individuals from the Cinnamomum camphora species to detect the polymorphism of 74 pairs of SSR primers that can be stably amplified, and finally obtain 21 pairs of transcriptome SSR polymorphic primers; then, select polymorphisms that perform well, and Seven pairs of SSR primers with clear bands were used for PCR amplification of 11 camphor species. POPGENE was used to calculate the relevant genetic parameters, and based on the PIC value (polymorphic information content), Shannon diversity index and Na value, four pairs of primers with stable amplification, good polymorphism and high repeatability were finally select...
Embodiment 3
[0045] Application Example 1 The method for quickly identifying the camphor species of the camphor species molecular detection primer composition comprises the following steps:
[0046] 1) The DNA extraction of the camphor species to be tested was completed by using the Ezup Column Plant Genomic DNA Extraction Kit from Shanghai Sangon Bioengineering Co., Ltd.
[0047] 2) Carry out PCR amplification sequentially according to the primer sequence in Table 1 according to the following PCR reaction system and reaction program until the identification is completed.
[0048] PCR reaction system 10 μL, including template DNA 0.6 μL (30ng), 10×Buffer 1 μL, 2.5mM dNTPs 0.1 μL (TAKARA company), 25mM MgCl 2 0.3 μL, 10 μM F-primer 0.4 μL (Nanjing GenScript Biotechnology Co., Ltd.), 10 μM R-primer 0.4 μL (Nanjing GenScript Biotechnology Co., Ltd.), Taq enzyme 0.2 μL (TAKARA Company), ddH 2 07 μL;
[0049] The PCR reaction program was: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C ...
Embodiment 4
[0058] The remaining 3 pairs of primers were selected from the SSR primers with good polymorphism and clear bands developed in Example 1. The specific primer information is shown in Table 4, and 11 varieties were identified. The identification method was the same as in Example 3.
[0059] Table 4 Information of the remaining 3 pairs of primers
[0060]
[0061]
[0062] As shown in Table 5 of the identification results, the selected 3 pairs of primers could not distinguish each variety, and the identification work could not be completed.
[0063] Table 5 utilizes the remaining 3 pairs of primers to identify the results of 11 varieties
[0064] breed name Cc_eSSR5 Cc_eSSR6 Cc_eSSR7 Gantong No.1 BB AB Gantong No. 2 BB BB AB Gantong No. 3 BB BB AAA Gantong No.4 BB BB AB Gantong No. 5 BB BB AB Gantong No. 6 BB BB AB Gantong No. 7 EE AB AB Gantong No. 8 DE BB BB Xiagua...
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