SSR (simple sequence repeat) molecular marker of akebia trifoliate as well as application and preparation method of SSR molecular marker
A molecular marker, standard technology, applied in the field of genetic engineering, to achieve the effect of high speed, high efficiency and low cost
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Embodiment 1
[0103] (1) Determine the concentration, purity, and sequencing depth of sample RNA to ensure the density and efficiency of molecular marker development, so as to ensure that the expected experimental purpose is achieved.
[0104]The total RNA of August melon A68 sample was extracted by Trizol method, the purity and integrity of DNA were detected by 1.2% agarose gel electrophoresis, and the concentration was detected by NanoDrop-2000C ultramicro spectrophotometer (Thermo Fisher Scientific, USA). Analyze whether the obtained RNA meets the experimental standards of RNA-Seq (total amount ≥ 20ng, mass concentration ≥ 400ng / μL, OD260 / 280 range between 1.8-2.2, RNA integrity number value ≥ 8, 28S / 18S ≥ 1.0).
[0105] (2) Construction and sequencing of cDNA library
[0106] Use Oligo(dT) to adsorb and purify mRNA, break mRNA into short fragments, use fragmented mRNA as a template, use six-base random primers to synthesize first-strand cDNA, and then add buffer, dNTPs, DNApolymerase, e...
Embodiment 2 8
[0121] Example 2 Analysis of Genetic Diversity of August Melon Germplasm Resources
[0122] 1 Materials and methods
[0123] 1.1 Materials
[0124] Eighty-eight August melon germplasm resources from different regions, resource names are shown in Table 3.
[0125] Table 3 Names of germplasm resources of August melon
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[0129] 1.2 Method
[0130] 1.2.1 Genomic DNA extraction
[0131] Collect 88 young leaves of August melon germplasm resources, and use Tiangen DNA kit to extract DNA. After the concentration of the extracted DNA is detected by electrophoresis, the concentration of the sample DNA is calculated and diluted to the required concentration.
[0132] 1.2.2 PCR using the designed SSR primers
[0133] PCR reaction system: 20μL reaction system composition is shown in Table 4:
[0134] Table 4 PCR reaction system
[0135] System composition
Final concentration
Magnesium ions (Mg 2+ )
2.0mmol / L
Taq bu...
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