A kind of est-ssr marker specific primer and screening method for the transcriptome sequence of Torreya torreya
A transcriptome sequence and specific primer technology, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection, etc., can solve the problems of complex steps, high development cost, and no EST-SSR marker research report.
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Embodiment 1
[0138] 1. Source of SSR loci and screening of EST-SSR sequences
[0139] The inventors used Trinity software to assemble the transcriptome data of the small torreya seed kernel transcriptome data and the round torreya seed kernel transcriptome data in the laboratory, and obtained the transcript sequence. A total of 246862 Transcripts and 142213 Unigenes were assembled. Use the MISA program (MIcroSAtellite identification tool, http: / / pgrc.ipk-gatersleben.de / misa / misa.html) to search for the SSR site of the Unigene above 1 kb obtained through screening. For the microsatellite fragments whose repetition times are 9, 8, 5, 5, and 5 times in sequence for repeating units of 2-6 bases, ESTs sequences containing microsatellite repeats are obtained. Using the primer design software Primer3.0 to design specific primers on the flanking sequences at both ends of the microsatellites.
[0140] 2. Design of EST-SSR labeled primers
[0141] In the flanking sequence of the microsatellite re...
Embodiment 2
[0150] 1. Extract the DNA of the Torreya tree
[0151] Specific steps are as follows:
[0152]Put CTAB in a 65°C water bath for 30 minutes in advance, take a fresh sample with an ice box, cut it into pieces and put it in a 2ml centrifuge tube (the sample volume is about 1 / 4 of the tube). Add a steel ball with a diameter of about 4mm into each centrifuge tube, soak it in liquid nitrogen for 5-7 minutes, fully pre-cool it, and grind it with a homogenizer. Add 800 μl of CTAB extract that has been water-bathed in advance to the centrifuge tube (add 1% β-mercaptoethanol to the CTAB extract), place the centrifuge tube in a water bath at 65°C for 40 min, and shake it upside down once during the period. Centrifuge at 12000rpm at 4°C for 10min, take the supernatant, add an equal volume of 24:1 chloroform-isoamyl alcohol (chloroform:isoamyl alcohol=24:1), shake well, and let stand for 5-8min. 12000rpm, 4℃, centrifuge for 10min, take the supernatant, add an equal volume of 24:1, shake ...
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