A method for developing ssr primers for the endangered sheep azalea based on rad-seq

A technology of sheep deer and genome, applied in the fields of molecular biology and bioinformatics, can solve the problems of low development efficiency, high development cost, high technical requirements, etc., and achieve the effect of quick cost and high efficiency of the method

Active Publication Date: 2018-08-10
JIANGXI NORMAL UNIV
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Problems solved by technology

The process of developing SSR tags by this method is very complicated, the technical requirements are high, and the development efficiency is also very low. Often only a small number of SSR tags can be developed, and the development cost is very high, etc.

Method used

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  • A method for developing ssr primers for the endangered sheep azalea based on rad-seq
  • A method for developing ssr primers for the endangered sheep azalea based on rad-seq
  • A method for developing ssr primers for the endangered sheep azalea based on rad-seq

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Embodiment Construction

[0021] The present invention is achieved like this, and described method comprises the following steps:

[0022] 1. DNA extraction and quality inspection: The modified CTAB method was used to extract the genomic DNA of the roe deer, the purity and integrity of the DNA were detected by agarose gel electrophoresis and Nanodrop, and the concentration of the genomic DNA was detected by Qubit.

[0023] 2. Library construction: Qualified DNA samples are digested, randomly interrupted, end-repaired, A-tailed, sequenced adapters added, purified, PCR amplified, and 300-700bp sequences are recovered by agarose gel electrophoresis, etc. That is, the genome library preparation is completed. The schematic diagram of library construction is attached figure 1 .

[0024] 3. Library inspection: After the library is constructed, use Qubit2.0 for preliminary quantification, dilute the library to 1ng / ul, then use Agilent 2100 to detect the insert size, and then use the Q-PCR method to accuratel...

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Abstract

The present invention provides a method for developing SSR primers for endangered sheep roe deer based on RAD-seq. The present invention includes constructing a genome library of the roe deer, obtaining Raw Data 7.653G after double-end sequencing and subsequent processing, and filtering Clean data 7.513G; using cd-hit-est clustering software to perform reads containing enzyme recognition sites Clustering, gather together the consistent regions captured by Reads2 during RAD sequencing; a maximum of 3 base mismatches are allowed between Reads2, and the reads with similar RAD-tags are clustered into one class through the sequence similarity of Reads2, using VelvetOptimiser The assembly software performs partial assembly of each type of sequencing data after screening, and finally selects the best congtig for SSR retrieval; after contig filtering, a total of 11,961 SSR fragments can be used to design SSR primers; use Primer3 to design SSR primers , 11,687 pairs of SSR primers were successfully designed by applying this method; 60 pairs of primers were randomly selected from them to detect polymorphisms in 6 DNAs from different geographic distributions of roe deer. It provides a new idea for the development of ssR primers for the sheep roe.

Description

technical field [0001] The present invention relates to molecular biology and bioinformatics, and relates to the development of endangered sheep deer (Rhododendron molle G. Don) SSR primer method. Background technique [0002] Rhododendron molle (Rhododendron molle G.Don), also known as Naoyanghua, Yangbubivorous, Yellow Rhododendron and Bacmima, is the only native species in the subgenus Rhododendron molle in my country. It is an important gene bank and an excellent parent for the improvement of rhododendron varieties in the world. At the same time, the whole plant organs of the sheep's hemp have important medicinal value. . [0003] Before the 1980s, sheep roe spread all over central and southern China. However, due to years of indiscriminate digging and habitat destruction, the reproductive link has been affected, and the area and population of sheep's roe in the wild have decreased sharply, and it is difficult to see it in the wild in some provinces. The sheep roe de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G06F19/22G06F19/20C12Q1/68C12N15/11
Inventor 罗向东程马龙戴亮芳周艳超谢建坤
Owner JIANGXI NORMAL UNIV
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