Method for obtaining SSR (Simple Sequence Repeat) primer of lycium ruthenicum based on transcription sequencing
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A technology of Lycium nigra and transcriptome sequencing, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of the lack of genome information and the small number of SSR primers, etc.
Active Publication Date: 2017-05-31
BEIJING FORESTRY UNIVERSITY
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[0006] At present, there is no genome information of Lycium
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Embodiment 1
[0078] A method for obtaining SSR primers of Lycium barbarum lycium barbarum based on transcriptional sequencing, the steps are as follows:
[0079] 1) Take the black fruit Lycium barbarum from 3 provenances as samples, and perform transcriptome sequencing on the above-ground parts, and divide them into control group, salt treatment group and mixed salt treatment group. The salt treatment group uses 100mL, pH=7, 200mmol / L NaCl solution, mixed salt treatment group used 100mL, pH=8.54, NaCl 2 CO 3 , NaCl, Na 2 SO 4 , NaHCO 3 According to the mixed solution with a final concentration of 200mmol / L mixed according to 1:9:9:1, after 6 hours of treatment, the leaves were frozen and stored in liquid nitrogen for transcriptome sequencing;
[0080] 2) Extract the total RNA of Lycium barbarum by CTAB method, separate the mRNA, combine it into double-stranded cDNA, add adenine and sequence linker, amplify the fragments with a size of 200bp-700bp, and use Illumina HiSeq 2000 to analyz...
Embodiment 2
[0100] Example 2 Utilize SSR primers to identify Lycium barbarum
[0101] 1. DNA extraction
[0102] Using the CTAB method to extract the DNA of 10 provenances of Lycium barbarum black fruit, 25 samples were taken from each provenance as repetitions, the specific steps are as follows:
[0103] ①Take the leaf sample stored at ultra-low temperature, grind it in a mortar and add it to a 2.0ml centrifuge tube (the amount of leaf powder added should not exceed the tip of the bottom of the 2.0ml centrifuge tube;
[0104] ②Quickly add 500 μl of CTAB preheated at 65°C (2% CTAB, add 0.5-1% β-mercaptoethanol before use), mix well, put it in a water bath at 65°C for half an hour, and shake it every ten minutes to make CTAB and leaves Mix the sample thoroughly;
[0105] ③Take out the centrifuge tube and let it stand for 2 minutes, add an equal volume of chloroform / isoamyl alcohol (24:1) mixture, mix the mixture with CTAB, and vortex to mix, so that CTAB and chloroform can fully contact;...
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Abstract
The invention relates to a method for obtaining an SSR (Simple Sequence Repeat) primer of lycium ruthenicum based on transcription sequencing, and belongs to the technical field of molecular marking techniques of molecular genetics of biology. The method comprises the following steps of (1), carrying out transcriptome sequencing; (2), screening an EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) site; (3), carrying out primer design; (4), extracting DNA (Deoxyribonucleic Acid); (5), carrying out primer screening; (6), carrying out polymorphic primer screening by using 8-percent native polyacrylamide gel electrophoresis. The method for obtaining the SSR primer of the lycium ruthenicum based on the transcription sequencing, which is provided by the invention, can be used as a method for the correction of a linkage map of a genome between remote species and comparative mapping, and has higher utilization value in two aspects. The greatest advantages of the method are that the development is simple and quick and the expense is low. The general characteristic of the SSR of the lycium ruthenicum is specified by utilizing an SSR molecular marking technique; the SSR primer of the lycium ruthenicum is developed; the foundation is laid for carrying out the research on the genetic diversity, the linkage map construction and the affinity relationship of species resources of the lycium ruthenicum by utilizing an SSR molecular marker, so that the method is better applied to molecular breeding.
Description
technical field [0001] The invention relates to a method for obtaining SSR primers, in particular to a method for obtaining SSR primers for Lycium barbarum lycium barbarum based on transcriptional sequencing, and belongs to the technical field of biological molecular genetic molecular markers. Background technique [0002] Simple sequence repeats (simple sequence repeats, SSR), also known as microsatellite DNA (Microsatellite DNA), refer to a class of DNA sequences composed of several (mostly 1 to 6) bases that are repeated in tandem. Repeats such as (AT)n, (GCA)n, (AATC)n, etc., are widely and evenly distributed in eukaryotic genomes. Due to the advantages of high polymorphism, good technical repeatability, and easy operation, SSR markers have been widely used as an excellent molecular marker in the genetic analysis of species since the early 1990s. People have done a lot of research on the occurrence and distribution of microsatellite DNA in humans, animals and plants. T...
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