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Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers

A microsatellite, curly hair technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA preparation, etc., can solve the problem of reducing heterozygosity, insufficient to complete small-scale spatial genetic structure research, SSR sequence polymorphism It can improve the accuracy, eliminate the interference of invalid alleles, and eliminate the tedious steps.

Inactive Publication Date: 2016-09-28
CHINA ACAD OF SCI NORTHWEST HIGHLAND BIOLOGY INST
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Problems solved by technology

However, the currently widely used microsatellite data analysis method (analysis of electrophoretic bands) is very likely to be interfered by null alleles (Nulllalleles) to reduce heterozygosity, and direct sequencing using microsatellite PCR amplification results is a very good solution
Although, previous studies have developed EST-SSR primers for this species, but the SSR sequence polymorphism in the coding region is low, which is not enough to complete the study of small-scale spatial genetic structure

Method used

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  • Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers
  • Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers
  • Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers

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Embodiment

[0053] Embodiment Twelve pairs of microsatellite primers for Chrysanthemum chrysophyllum microsatellite primers, amplification and sequencing methods include the following steps:

[0054] (1) Genomic DNA of the C. chrysogenum population of 63 individuals of the following three flora was extracted using the improved CTAB method (see Table 1).

[0055] Table 1 Chrysanthemum viridans ( F. luteovirens ) sample collection information

[0056]

[0057] Specific steps are as follows:

[0058] ① Weigh 0.5 g of the internal tissue at the junction of the fruit body stipe and the cap after drying in an oven at 45°C, add liquid nitrogen to the mortar for precooling, add 0.2 g of PVP powder, and put the material into the liquid Grind evenly in nitrogen until all are ground to powder;

[0059] ② Transfer to a 1.5 ml centrifuge tube, add 600 μL BI solution preheated at 65°C, shake in a water bath at 65°C for 10 minutes, centrifuge at 10,000 rpm at 4°C for 10 minutes, and discard the s...

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Abstract

The invention relates to a method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers. The method comprises the following steps: (1) extracting genome DNA of three floccularia luteovirens populations among which the geographic interval is more than 300km by using an improved CTAB method; (2) randomly selecting an individual genome DNA respectively from the three populations, mixing, detecting the quality of total DNA, preparing a gene library, and performing Illumina HiSeq<TM>2500 sequencing after the gene library is qualified in examination in depot; (3) splicing sequenced data, detecting simple sequence repeats (SSR) in the total DNA sequence by using SR search software, and performing primer design by applying primer3; (4) preparing an SSR primer having an annealing temperature of 50-60 DEG C by adopting a temperature gradient method; (5) respectively performing PCR amplification on the genome DNA of the three floccularia luteovirens populations, and sequencing and verifying to obtain 12 pairs of primers with polymorphism; and (6) calculating the number N of allelic genes, haplotype diversity H<d> genetic differentiation coefficient F<ST>, nucleotide diversity P, G<ST> and the value of pi. The method is beneficial to large-scale research.

Description

technical field [0001] The invention relates to the technical field of small-scale community structure and genetic diversity of fungi, and in particular to methods for designing, amplifying and sequencing twelve pairs of microsatellite primers for Cinnamomum chrysophylla. Background technique [0002] Chrysanthemum viridis ( Floccularia luteovirens ) belonging to the family Agaricales (Agaricales) T richolomataceae), is a fruiting body-producing ectomycorrhizal fungus mainly distributed in the Qinghai-Tibet Plateau. Because of its bright yellow color, delicious taste and rich nutrition, it has been regarded as a special delicacy on the Qinghai-Tibet Plateau for a long time and has high economic value. [0003] At present, humans lack the necessary understanding of this fungus, which is widely distributed in the Qinghai-Tibet Plateau and plays an important role in the fragile alpine meadow ecosystem, such as its reproductive mode, distribution and small-scale spatial geneti...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6869C12Q1/6806C12Q2525/151
Inventor 邢睿高庆波张发起陈世龙
Owner CHINA ACAD OF SCI NORTHWEST HIGHLAND BIOLOGY INST
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