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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

Active Publication Date: 2019-03-15
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the development of August melon SSR markers and the development of SSR markers using transcriptome technology have not been reported

Method used

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  • SSR (simple sequence repeat) molecular marker of akebia trifoliate as well as application and preparation method of SSR molecular marker
  • SSR (simple sequence repeat) molecular marker of akebia trifoliate as well as application and preparation method of SSR molecular marker
  • SSR (simple sequence repeat) molecular marker of akebia trifoliate as well as application and preparation method of SSR molecular marker

Examples

Experimental program
Comparison scheme
Effect test

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

[0126]

[0127]

[0128]

[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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Abstract

The invention relates to the technical field of gene engineering, in particular to an SSR (simple sequence repeat) molecular marker of akebia trifoliate as well as an application and a preparation method of the SSR molecular marker. Primers of the SSR molecular marker comprise one or more pairs of primers shown in sequences SEQ ID NO:1-98. The developed transcriptome SSR molecular marker of the akebia trifoliate can be used for analyzing genetic diversity of akebia trifoliate germplasm resources and lays a good theoretical foundation for molecular biology and genetic research of the akebia trifoliate. The SSR marker developed with a transcriptome high-throughput technology is high in speed, low in cost and high in efficiency.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the SSR molecular marker of August melon and its application and preparation method. Background technique [0002] August melon, scientific name Akebia trifoliate (Thunb.)], also known as Akebia trifoliate (Thunb.), also known as Akebia trifoliate, August fried, etc., is mainly distributed in the south of my country, the Yangtze River Basin and the southwest mountainous area. Ecologically pollution-free natural green fruit. August melon is rich in polyphenols, flavonoids, oleanolic acid, ursolic acid, amino acids, vitamins, minerals and other ingredients. Moreover, the active ingredients in the fruit have inhibitory effects on the malignant proliferation of various tumor cells. The oil content of its seeds is as high as more than 40%, which is a very high-quality natural high-grade vegetable edible oil. Therefore, August melon is a kind of treasure in the fruit that...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6811C12N15/11
CPCC12Q1/6811C12Q1/6895C12Q2600/156C12Q2535/122C12Q2537/165
Inventor 栾明宝牛娟陈建华王晓飞孙志民
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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