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STR molecule marker based on sika deer whole genome developing and application of STR molecule marker

A molecular marker, whole-genome technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., to reduce costs, improve accuracy, and facilitate genotyping

Active Publication Date: 2017-11-17
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is simpler and more convenient than the above methods, but it can only be applied to species with known sequence information such as rice and Arabidopsis. At present, this method is mainly used for the development of EST-SSR

Method used

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  • STR molecule marker based on sika deer whole genome developing and application of STR molecule marker
  • STR molecule marker based on sika deer whole genome developing and application of STR molecule marker
  • STR molecule marker based on sika deer whole genome developing and application of STR molecule marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 STR site screening and polymorphism analysis of 4-base repeat unit in sika deer

[0073] According to the results of sika deer genome sequencing and sika deer population resequencing, MISA analysis was first used to obtain 940,413 STR sites in the reference genome, and 14,863 STR sites containing 4-base repeating units were extracted; then searched the database for STR site polymorphisms There are 386 STR intervals containing InDels with a screening threshold greater than 7000.

[0074] A two-step method can be used to quickly screen the STR sites of highly polymorphic 4-base repeat units in the sika deer genome.

Embodiment 2

[0075] Example 2 Batch design of STR primers

[0076] Use Primer3 to design primers in batches for the screened STR sites. The primer length is limited to 20bp±2bp, the annealing temperature is controlled between 55°C-60°C, and the CG% is controlled between 30%-80%. Elimination is prone to mismatches And primers with hairpin structure, the product fragment size is controlled between 80-300bp. 284 pairs of STR primers were designed for 386 high-length polymorphic sites, and e-PCR was performed with the designed primers to remove non-specific amplification. Through comparative analysis in NCBI, try to make the screened STR markers cover all chromosomes , to avoid being located at the end of the chromosome, select 100 pairs of primers for subsequent polymorphism verification.

[0077] The Primer3 was used to design primers in batches, and the designed primers were screened and verified by e-PCR, and further screened by sequence comparison in the NCBI database. The developm...

Embodiment 3

[0078] Example 3 Polymorphism and authenticity verification of STR loci

[0079] The DNA of 8 unrelated sika deer individuals was extracted, and the extracted sika deer genomic DNA was used as a template for PCR amplification, and the primers were synthesized by Shanghai Sangong Biotechnology Company. A polyacrylamide gel was prepared, the amplified products were detected by electrophoresis and silver staining, and 31 pairs of primers with high polymorphism were screened according to the electrophoresis bands for subsequent sequencing verification. The PCR products amplified by 31 pairs of primers were sequenced, compared with the e-PCR amplification sequence using the reference genome as a template, and through consistency analysis, it was determined that all 31 STR sites were real polymorphic sites .

[0080] Through polyacrylamide gel detection and sequencing analysis, all 31 STR sites were real polymorphic sites.

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Abstract

The invention discloses an STR molecule marker based on sika deer whole genome developing and application of the STR molecule marker. Based on a sika deer whole genome sequence, screening for STR loci is conducted, primer is designed and the application effect is verified, and the sika deer STR molecule marker with high amplification efficiency and the high recognition rate is finally obtained and used for amplifying primer sequences of the STR molecule marker as shown in SEQ ID NO:1-62. According to the STR molecule marker based on sika deer whole genome developing and application of the STR molecule marker, a four-base repeated unit is contained in the sika deer STR locus, compared with routine two-base to three-base repeated units, the distinguishing degree is higher and more stable, gene type judging is easier, and the convenient and efficient molecule marker can be supplied for genetic research on sika deer groups.

Description

technical field [0001] The invention relates to the fields of animal resource science, molecular biology and bioinformatics, in particular to the STR molecular marker developed based on the whole genome of sika deer and its application. Background technique [0002] With the completion of various mammalian genome projects and the rapid development of sequencing technology and bioinformatics analysis technology, people's attention is increasingly focused on the evaluation, protection and utilization of special economic animal genetic resources, especially the genetic resources of sika deer. resource. Due to the special medicinal value of wild sika deer, my country began to domesticate and utilize wild sika deer from the 14th to 12th centuries BC. It is mainly used as raw material of traditional Chinese medicine and health products. At present, the wild sika deer in my country is very rare, and only exists in some fragmented habitats, and its distribution area is shrinking. T...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6888C12Q2600/156
Inventor 邢秀梅胡鹏飞徐佳萍刘华淼张正义
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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