Method for developing ramie genome SNP marks on large scale and primer developed by method
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A genome and large-scale technology, applied in the field of genetic engineering, can solve problems such as ramie SNP markers that have not yet been developed, and achieve the effect of low cost, high efficiency and fast speed
Inactive Publication Date: 2014-12-17
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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At present, there is no relevant report on the method of developing ramie SNP markers
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Embodiment 1
[0019] (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.
[0020] The DNA of Ramie No.1 and Hejiang Qingma was extracted.
[0021] Enzyme digestion scheme selection
[0022] According to the selection principle of closely related species, the cannabis genome was finally selected as the reference genome for enzyme digestion prediction.
[0023] Ramie species information and related species information are as follows:
[0024] Ramie information: Genome size is 716Mb, GC content is 49%;
[0025] Related species cannabis information: Genome size is 757Mb, GC content is 24%, download address:
[0061] The new shoots of 4 ramie germplasms (Zhongzhu No. 1, Hejiang Qingma, Zhongzhu No. 2, Hongpi Honghua) were taken from the National Germplasm Changsha Ramie Garden, and DNA was extracted 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.
[0062] 1.2.2 PCR using the designed SNP primers
[0063] PCR reaction system: 20μL reaction system composition is shown in Table 4
[0064] Table 4 Composition of PCR reaction system
[0065] System composition
Final concentration
Mg 2+
2.0mmol / L
Taq Buffer
1×
dNTP Mix
200μmol / L each
...
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Abstract
The invention discloses a method for developing ramie genome SNP marks on a large scale and a primer developed by the method. The method particularly comprises the following steps: (1) extracting DNAs of two ramie varieties; (2) carrying out enzyme digestion on two ramie variety genomes by using RsaI enzyme, and thus obtaining small fragments of 214-314bp; (3) sequencing the small fragments obtained by enzyme digestion, and thus obtaining SLAF labels; (4) carrying out polymorphic analysis as to the SLAF labels obtained by the two ramie varieties according to the number of alleles and the difference between the gene sequences, and thus searching the SLAF labels with SNP; and (5) carrying out primer design on the searched SLAF labels, and detecting to be genome SNP marks. Compared with a traditional method for developing molecular markers, the method is feasible, simple and convenient to operate, high in efficiency, and low in cost, and a solid foundation is established for ramie molecular biology and genetics by obtaining of the ramie genome SNP marks on a large scale.
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 SNP markers from ramie genome and primers developed therefor. Background technique [0002] Among many molecular markers, SNP markers are currently the most studied and most promising molecular markers. (Tang Liqun et al., Application and Research Progress of SNP Molecular Markers. China Agricultural Science Bulletin (28): 154-158.) Compared with other molecular marker technologies, SNP markers have a large number of genomes, wide distribution and do not need to be based on the size of DNA fragments. Banding DNA can realize large-scale and automation, so it is more suitable for a large number of genetic detection and analysis (Liu Chuanguang et al., Single Nucleotide Polymorphism in Rice and Its Application. Genetics, 2006(28): 737-744 .). Therefore, it has been paid attention to in many aspects of plant genetic re...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 陈建华栾明宝王晓飞许英孙志民刘晨晨
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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