SNP (single nucleotide polymorphism) markers based on KASP (kompetitive allele-specific polymerase chain reaction) and application of SNP markers

A marker and genome technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, microbial determination / inspection, etc., can solve the problems of cigar resource identification and variety approval that have not yet been seen with SNP molecular markers

Active Publication Date: 2020-06-30
中国烟草总公司海南省公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

In recent years, SNP markers have been widely used in the fingerprint construction and genetic evolution analysis of wheat, rice, corn and other stap

Method used

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  • SNP (single nucleotide polymorphism) markers based on KASP (kompetitive allele-specific polymerase chain reaction) and application of SNP markers
  • SNP (single nucleotide polymorphism) markers based on KASP (kompetitive allele-specific polymerase chain reaction) and application of SNP markers
  • SNP (single nucleotide polymorphism) markers based on KASP (kompetitive allele-specific polymerase chain reaction) and application of SNP markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0416] Screening of cigar core SNP markers

[0417] In the early stage, 113 cigar resources were simplified genome sequencing. GBS (Genotyping-by-sequencing) technology refers to genotyping by sequencing, and constructing SNP molecular markers by selecting appropriate restriction enzymes combined with high-throughput population sequencing. To construct the GBS library, first use restriction endonuclease to digest the genomic DNA, then add a sequencing linker with barcode, mix the samples, construct a small fragment library (250-550bp), and perform PE125 paired-end sequencing.

[0418] 1. Genome electronic digestion evaluation

[0419] In order to check the efficiency of digestion and analyze all markers, the genome is digested according to the site of the restriction endonuclease to the reference genome, and the corresponding situation is counted, which is convenient for later evaluation. Effective represents the molecular markers that meet the criteria for screening fragment...

Embodiment 2

[0442] A method for identifying 111 cigar resources using 48 cigar core SNP markers

[0443] The 111 cigar germplasm resources are the cigar germplasm resources collected and preserved by the National Tobacco Germplasm Resources Medium-term Bank in recent years. Only phenotype identification has been carried out. If there are more consistent phenotypes, it is impossible to identify whether they are the same cigar resources. There is also the phenomenon that resource names are similar or even identical. Apply 48 cigar tobacco core SNP markers and KASP primers of the present invention to carry out genetic relationship identification to the above resources, and the specific experimental steps are as follows:

[0444] (1) Preparation of DNA template: 111 cigars were sampled at the stage of 5 true leaves, and genomic DNA was extracted by CTAB method.

[0445] (2) Design and synthesis of KASP primers: When designing KASP primers for SNP sites, it is necessary to add a FAM fluoresce...

Embodiment 3

[0456] A method for analyzing the population structure of cigar tobacco resources using cigar tobacco core SNP markers

[0457] When the present invention screens the core SNP markers of cigars, considering the polymorphism and specificity of the markers, the selected part comes from the information of specific SNP sites of different groups, so the cigars resource population structure analysis can be carried out. The 48 pairs of KASP markers developed in Example 1 were used to analyze the population structure of 111 cigar resources from all over the world. 111 cigar tobacco resources DNA extraction, machine reaction and subordinate genotype acquisition and result analysis, the specific methods are the same as in Example 2. Population genetic structure refers to a nonrandom distribution of genetic variation in a species or population. Individuals in the same subgroup have higher kinship, while the kinship between subgroups is slightly distant. Population structure analysis he...

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Abstract

The invention provides an SNP (single nucleotide polymorphism) markers based on a KASP (kompetitive allele-specific polymerase chain reaction) and application of the SNP markers and belongs to the technical field of molecular biology, germplasm resources and molecule breeding techniques. The SNP markers comprise 48 markers such as CigarSNP01-1, CigarSNP01-2 and CigarSNP02-1. By adopting the SNP markers provided by the invention, cigar SNP fingerprint spectrum construction can be completed, conventional cigar resources can be systematically identified, repeatedly collected resources can be alsorejected, and intellectual property protection mechanisms of cigar varieties can be constructed. The SNP markers provided by the invention are adopted to screen and identify collected cigar resourcesand examine and approve newly cultured cigar varieties, and are applied to cigar breeding operation.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology, germplasm resource science and molecular breeding, and in particular relates to a set of KASP-based SNP markers and applications thereof. Background technique [0002] Because of its unique style, profound cultural connotation and higher smoking safety, cigars attract more and more consumers. In recent years, domestic and foreign market demand has been increasing, and the industry has broad prospects. However, my country’s cigar research work started late, and basic research lagged behind. There are many cigar resources and varieties introduced, collected and exchanged by various research units. use. Moreover, the specific genetic background of cigars is not yet clear, and conventional breeding and molecular breeding of cigars cannot be carried out effectively and quickly. Therefore, constructing fingerprints for these resources, eliminating repeatedly collected and imported resources...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 刘国祥吕洪坤张兴伟李媛向小华刘好宝戴培刚佟英杨爱国冯全福
Owner 中国烟草总公司海南省公司
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