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Persimmon tree colletotrichum gloeosporioide SSR (Simple Sequence Repeat) primer pair developed based on sibling species genome and application of primer pair

An anthracnose fungus and primer pair technology is applied in the field of SSR primers, persimmon anthracnose fungus SSR primer pairs, and can solve the problems of strong randomness and poor stability.

Active Publication Date: 2017-05-24
河南省林业科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

AFLP, ISSR, RAPD and other markers all use markers without genome sequence information. Although they have certain practicability, they have strong randomness and poor stability

Method used

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  • Persimmon tree colletotrichum gloeosporioide SSR (Simple Sequence Repeat) primer pair developed based on sibling species genome and application of primer pair
  • Persimmon tree colletotrichum gloeosporioide SSR (Simple Sequence Repeat) primer pair developed based on sibling species genome and application of primer pair
  • Persimmon tree colletotrichum gloeosporioide SSR (Simple Sequence Repeat) primer pair developed based on sibling species genome and application of primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the acquisition of persimmon anthracnose bacteria

[0042] In 2014, the diseased leaves were collected from the main producing areas of persimmons, and the anthracnose fungus of persimmon tree was isolated by tissue separation method, and purified by isolation of single spores, and the fungus of anthracnose persimmon tree was obtained. The present invention has isolated 23 persimmon anthracnose bacteria (see Table 1).

[0043] The specific operation is as follows: collect the fallen persimmon leaves, cut off the small piece of diseased tissue (the junction of disease and health) of the leaves, put the diseased tissue in 75% alcohol for 2-3 seconds, remove it and put it into 5% sodium hypochlorite for sterilization 2-3 minutes (the branch tissue is large and rough, and it needs to be sterilized for 3-5 minutes to prevent contamination); then rinse it with sterile water three times; use sterilized absorbent paper to absorb the moisture of the diseased tissue...

Embodiment 2

[0046] Embodiment 2: the extraction of persimmon anthracnose bacteria DNA

[0047] The CTAB (cetyltrimethylammonium bromide) method was used to extract the genomic DNA of the persimmon anthracnose bacterium, and the specific operations were as follows:

[0048](1) Transfer the preserved persimmon anthracnose bacterial strain into liquid PDA and cultivate for 3 days, pick out the mycelium block and dry it with filter paper, put it into a mortar and add liquid nitrogen to grind it into powder, and obtain mycelium powder; Put 50mg of mycelium powder in a 1.5ml EP tube, add 900μl 2% CTAB (cetyltrimethylammonium bromide) extract and 90μl 10% SDS (sodium dodecylbenzenesulfonate), mix well and place Place in a water bath at 55-60°C for 1 hour; (2) Centrifuge at 12000rpm / min for 5min, take the supernatant, add an equal volume of phenol\chloroform\isoamyl alcohol (25:24:1) to extract once; (3) 12000rpm / Centrifuge for 5 min, absorb the supernatant, add an equal volume of chloroform to...

Embodiment 3

[0049] Example 3: Development of SSR primers

[0050] (1) Log in to the NCBI website, select the genome database, search for the genome data of anthracnose, find the complete genome of Colletotrichum gloeosporioides Nara gc5, a close relative of the persimmon anthracnose, and download the complete genome of the gloeosporioides Nara gc5 Data, including all scaffolds or contigs.

[0051] (2) Use MISA software to search for SSR sites on the whole genome sequence downloaded in step (1). The specific process is as follows: organize the genome data into FASTA format, download and install Perl language, and run the MISA program to identify and locate the genome sequence. For SSR, the parameters are set as follows: the number of repetitions of mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides is at least 10, 6, 5, 5, 5 and 5; after SSR site search, a total of 1981 mononucleotide repeat sequences, 1146 dinucleotide repeat sequences,...

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Abstract

The invention belongs to the technical field of biologics, and in particular discloses a persimmon tree colletotrichum gloeosporioide SSR (Simple Sequence Repeat) primer pair developed based on sibling species genome and application of the primer pair. According to the persimmon tree colletotrichum gloeosporioide SSR primer pair, 30 pairs of SSR primers are developed by finding SSR by using MISA software on the basis of the genome of a sibling species, namely, colletotrichum gloeosporioides, of persimmon tree colletotrichum gloeosporioides, and 15 pairs of polymorphism SSR primers are obtained by screening 6 different persimmon tree colletotrichum gloeosporioides; the persimmon tree colletotrichum gloeosporioide SSR primer pairs provided by the invention are novel markers which exist stably, can be used for analyzing genetic diversity and genetic differentiation on the persimmon tree colletotrichum gloeosporioides, can be also used for identifying varieties of the persimmon tree colletotrichum gloeosporioides and studying ties of consanguinity, and have very important significances in effective prevention and control on the persimmon tree colletotrichum gloeosporioides.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to SSR primers, in particular to a set of SSR primer pairs developed on the basis of genomes of related species and an application thereof. Background technique [0002] Persimmon has been cultivated in my country for more than 3,000 years. It is widely loved by people because of its delicious taste, high nutritional value and medicinal value. Anthracnose of persimmon tree is a very important disease on persimmon tree, which is caused by Colleotrichum horii. It occurs more seriously on Fupingjian persimmon and Guangxi water persimmon in my country, which has caused great economic losses to the development of persimmon industry in China. The year-by-year increase of persimmon anthracnose damage is closely related to the long-term co-evolution of the pathogen and persimmon trees, as well as the adaptation to the local climate and environment. An in-depth analysis of the genetic diversity a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q2600/156C12Q2600/16
Inventor 丁向阳崔林开徐建强邓全恩周耀伟胡清坡贺凡刘乃辉许延松李鑫鹏高地玲
Owner 河南省林业科学研究院
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