EST-SSR primer group developed based on radix achyranthis bidentatae transcriptome and acquisition method thereof
An acquisition method and transcriptome technology, applied in the field of molecular biology, can solve problems such as lack of specificity, lack of genetic basis of Achyranthes bidentata, inability to accurately evaluate the genetic background of Achyranthes bidentata germplasm resources, and achieve the effect of saving resources
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Embodiment 1
[0032] (1) Achyranthes bidentata transcriptome library constructed,
[0033] The roots of the "Huai Niu Xi No. 1" variety at the seed formation stage (repeated 3 times, a total of 6 samples) were collected for 1 year of planting (R1) and 2 years of continuous planting (R2), and the total RNA was extracted by the Trizol method, and the Magnetic beads with Oligo(dT) enrich mRNA. Add fragmentation buffer to fragment mRNA into short fragments, use mRNA as a template, use 6-base random primers (randomhexamers) to reverse transcribe to synthesize the first cDNA strand, and then add buffer, dNTPs, RNase H and DNA polymeraseI to synthesize double strands cDNA strands, and then use AMPure XP beads to purify double-stranded cDNA and add adapters. The cDNAs are first repaired, A-tailed and sequenced adapters are connected, and then AMPure XP beads are used for fragment size selection. Finally, PCR amplification was performed, and the PCR products were purified with AMPure XP beads, and ...
Embodiment 2
[0045] The impact on the PCR amplification product of EST-SSR was analyzed under different PCR treatment conditions. The experimental and analysis results are as follows:
[0046] (1) The PCR amplification reaction system is 20 μL, the concentration of Taq DNA polymerase (60 U / L), the concentration of forward and reverse primers (0.2 μmol / L), the concentration of dNTP (200 μmol / L), and the amount of template DNA set to 8 Gradients, respectively 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 mg / L, were carried out on an ABI 2720 PCR amplification instrument in the United States. The reaction program was: pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 40 seconds, 30 cycles; extension at 72°C for 5 minutes; detection by 1% agarose gel electrophoresis.
[0047] The result is as figure 2 As shown, the M lane is DL2000 Marker, and the Achyranthes knuckle DNA concentration can obtain specific and clea...
Embodiment 3
[0058] All primers in Table 1 were selected and designed using Primer3 software. Based on the Achyranthes knuckles EST-SSR amplification system optimized in Example 2, 22 EST-SSRs were randomly selected to verify the effectiveness of the established amplification system. Such as Figure 7 As shown, specific target bands were detected for 21 EST-SSR sequences, indicating that the PCR detection method for identifying EST-SSR sequences established by the present invention can be used for subsequent experiments in identifying EST-SSR markers of Achyranthes bidentata.
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