Method for developing endangered rhododendron molle SSR primer on basis of RAD-seq

A sheep slug and genome technology is applied in the field of developing endangered sheep slug SSR primers based on RAD-seq, which can solve the problems of low development efficiency, high development cost, complicated SSR labeling process, etc., and achieve the effect of fast cost and efficient method.

Active Publication Date: 2015-05-06
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

Method used

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  • Method for developing endangered rhododendron molle SSR primer on basis of RAD-seq
  • Method for developing endangered rhododendron molle SSR primer on basis of RAD-seq
  • Method for developing endangered rhododendron molle SSR primer on basis of 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 300-700 bp sequences recovered by agarose gel electrophoresis, etc. , which completes the genome library preparation. 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 accurately quan...

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Abstract

The invention provides a method for developing an endangered rhododendron molle SSR primer on the basis of RAD-seq. The method comprises the steps: establishing a rhododendron molle genomic library, acquiring 7.653G of Raw Data and 7.513G of filtered Clean data after the paired-end sequencing and subsequent treatment; clustering reads containing enzyme recognition sites by utilizing cd-hit-est clustering software, and gathering areas which are consistent in Reads2 capture when in RAD sequencing; at most mispairing three basic groups between Reads2, gathering the reads with similar RAD-tag by virtue of the sequence similarity of the Reads2, locally assembling each type of screened sequencing data by utilizing VelvetOptimiser assembling software, and selecting a best congtig to search SSR; collectively obtaining 11961 SSR fragments for designing the SSR primer after the contig is filtered; designing the SSR primer by utilizing Primer3, wherein 11687 pairs of SSR primers can be successfully designed by utilizing the method; randomly selecting 60 pairs of primers to carry out polymorphic detection for 6 parts of rhododendron molle DNAs in different geographical distribution. By adopting the method, a novel concept is provided for developing the rhododendron molle SSR primer.

Description

technical field [0001] The present invention relates to molecular biology and bioinformatics, and relates to the development of endangered sheep azalea ( Rhododendron molle G. Don) SSR primer method. Background technique [0002] Sheep roe ( Rhododendron molle G. Don), also known as Naoyanghua, Yangbugrass, Yellow Rhododendron, and Balima, is the only native species in the subgenus of Rhododendronaceae 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 deer...

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Application Information

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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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