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Method for identifying diospyros species based on SRAP (Sequence Related Amplified Polymorphism) molecular marker

A molecular marker, SRAP-PCR technology, applied in the field of primer combinations based on SRAP molecular markers to identify different species of Persimmon, achieving high stability, repeatability, and convenient operation

Inactive Publication Date: 2014-09-24
NORTHWEST A & F UNIV
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Problems solved by technology

[0004] At present, the traditional identification method based on morphology can no longer meet the requirements of effectively distinguishing or identifying many germplasm resources. The role of plant genetic diversity and species resource identification based on the DNA molecular level has been recognized. This technology overcomes the traditional morphological Factors that are susceptible to external influences on biological, cytological and biochemical markers

Method used

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  • Method for identifying diospyros species based on SRAP (Sequence Related Amplified Polymorphism) molecular marker
  • Method for identifying diospyros species based on SRAP (Sequence Related Amplified Polymorphism) molecular marker

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Embodiment 1

[0019] A method for identifying persimmon species based on SRAP molecular markers, the specific steps are as follows:

[0020] (1) Genomic DNA extraction:

[0021] The persimmon species resources in the embodiments of the present invention are all important persimmon germplasm resources, and these germplasm resources belonging to 5 species are all taken from the National Persimmon Germplasm Resource Garden.

[0022] The modified CTAB method was used to extract DNA from each germplasm resource. The specific steps were as follows: take about 0.2-0.5 g of persimmon leaves dehydrated by color-changing silica gel in a mortar, add liquid nitrogen to fully grind them, and then transfer them to a 2 mL centrifuge tube. Add 940uL preheated (CTAB buffer780uL, PVP140uL, β-mercaptoethanol20uL) extraction buffer, incubate at 65°C for 30min, and mix once every 5min (shake gently). Add 940 uL of chloroform-isoamyl alcohol (24:1), shake gently, and centrifuge at 10000 rpm at 4°C for 10 min. ...

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Abstract

The invention discloses a method for identifying diospyros species based on an SRAP (Sequence Related Amplified Polymorphism) molecular marker. A primer for identifying diospyros species (varieties) is a primer combination formed by screening and verifying 357 pairs of primers in a large scale, wherein the primer combination comprises an upstream primer and a downstream primer respectively having the concrete nucleotide sequences as follows: the nucleotide sequence of the upstream primer is TGA GTC CAA ACC GGACA, and the nucleotide sequence of the downstream primer is GAC TGC GTA CGA ATT CAA. The SRAP primer for rapidly distinguishing diospyros species is obtained by strictly screening in a large scale so as to have relatively high stability and repeatability; five diospyros species (varieties) can be easily distinguished through one pair of primers and one PCR (Polymerase Chain Reaction), so that the method is convenient to operate, rapid and accurate; the method disclosed by the invention can be used for rapidly and accurately distinguishing the five diospyros species under any condition, and can also be used for realizing the taxonomic identification of wild diospyros resources by combining a morphological feature.

Description

technical field [0001] The invention relates to the field of molecular biology molecular markers, in particular to a combination of primers and a method for identifying different species (variants) of the persimmon genus based on SRAP molecular markers. Background technique [0002] Persimmon (D.kakiThunb., 2n=6x=90) is a kind of persimmon plant of Persimmonaceae. It is found in the south of 41° north latitude in my country, Wushan, Tianshui in Gansu, Emeishan, Daliangshan and Yunnan in Sichuan, especially in the Yellow River Basin. It is widely planted in Shandong, Hebei, Henan, Shanxi, and Shaanxi provinces. Persimmon originated in my country and has a cultivation history of more than 3,000 years in my country. In addition to the persimmon species, there are 56 species (variants) of the persimmon genus in my country. Among them, the wild persimmon is a variant of the persimmon species. A new species discovered, in addition to more commonly used Zhejiang persimmon, oil pers...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2565/125
Inventor 杨勇井赵斌阮小凤夏乐晗杨婷婷张永芳王仁梓
Owner NORTHWEST A & F UNIV
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