A method for developing ramie genome ssr markers in large quantities and the primers developed therefor
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A genome, large-scale technology, applied in the field of genetic engineering, to achieve the effect of high cost, high efficiency and low cost
Inactive Publication Date: 2016-08-24
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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Problems solved by technology
At present, the SSR markers developed by Ramie mainly use the magnetic bead enrichment method and the development of SSR markers from EST, and the developed SSR markers are less than 2000 pairs
The development of SSR markers using SLAF-seq technology has not been reported
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Embodiment 1
[0022] (1) Determine the enzyme digestion plan, gel cutting range, sequencing volume, etc. to ensure the density and uniformity of molecular marker development to ensure that the expected experimental purpose is achieved.
[0023] The DNA of Ramie No.1 and Hejiang Qingma was extracted.
[0024] Enzyme digestion scheme selection
[0025] According to the selection principle of closely related species, the cannabis genome was finally selected as the reference genome for enzyme digestion prediction.
[0026] Ramie species information and related species information are as follows:
[0027] Ramie information: Genome size is 716Mb, GC content is 49%;
[0028] Related species cannabis information: Genome size is 757Mb, GC content is 24%, download link:
[0031] The cannabis genome was systematically analyzed using enzyme digesti...
Embodiment 2
[0058] Primer polymorphism detection
[0059] 1 Materials and methods
[0060] 1.1 Materials
[0061] 24 selfed offspring of Zhongzhu 1;
[0062] There are 24 ramie varieties, and the variety names are shown in Table 4.
[0063]
[0064] 1.2 Method
[0065] 1.2.1 Genomic DNA extraction
[0066] DNA was extracted from 24 selfed offspring of Zhongzhu No. 1 and new shoots from 24 individual plants of ramie varieties using the Tiangen kit. After the concentration of the extracted DNA is detected by electrophoresis, the concentration of the sample DNA is calculated and diluted to the required concentration.
[0067] 1.2.2 PCR using the designed SSR primers
[0068] PCR reaction system: 20μL reaction system composition is shown in Table 5
[0069] Table 5 PCR reaction system composition
[0070] System composition
Final concentration
Mg 2+
2.0mmol / L
Taq Buffer
1×
dNTP Mix
200μmol / L each
Taq Enzymes
1U
Primers...
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Abstract
The invention provides a method for large-scale development of ramie genome SSR (simple sequence repeat) markers and primers developed by the method. The method comprises the following concrete steps: (1) extracting ramie DNA; (2) performing enzyme digestion on ramie genomes by virtue of RsaI enzyme to obtain small fragments of 214-314bp; (3) sequencing the small fragments obtained by enzyme digestion to obtain SLAF labels; (4) searching SSR sequences by virtue of SSR hunter for the labels; and (5) performing primer design on the searched SSR sequences, and performing polymorphism detection on primers to obtain the polymorphism primers-ramie genome SSR markers. Compared with a conventional magnetic bead enrichment method, the method is feasible, simple and convenient to operate, high in efficient and low in cost; the acquisition of the large-scale ramie genome SSR markers lays a solid foundation for molecular biology and genetics of ramie.
Description
technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a high-throughput method for developing SSR markers from ramie genome and primers developed therefor. Background technique [0002] Simple sequence repeat (SSR), also known as microsatellite (microsatellite), refers to a DNA sequence repeated in tandem multiple times in the genome in units of 1 to 6 nucleotides (Akkaya M, Bhagwata A, Cregan B .1992. Length polymorphisms of simple repeat DNA insoybean. Genetics. 132:1131-1139). Compared with other molecular marker technologies, SSR markers have the advantages of easy detection, co-dominant inheritance, good repeatability, abundant quantity and high polymorphism, and spread throughout the genome, so they have been valued in many aspects of plant genetic research (Schlotterer C .2004.The evolution of molecular markers-justa matter offashion.Nat Rev Genet.5:63-69). SSR can be divided into genomic SSR and EST...
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