Method of developing polymorphic EST-SSR marker by utilizing complete genome and EST data

Abstract

The invention discloses a method of developing a polymorphic EST-SSR marker by utilizing a complete genome and EST data and belongs to the field of molecular biology. The method comprises the following steps: acquiring a genomic sequence and the EST data firstly; then searching an SSR site in the complete genome, and identifying and screening a single SSR site in the complete genome; then designing a comparison primer of the single SSR site, and carrying out comparison with EST sequence data as a template; counting a comparison result, and screening the SSR site with more than two stimulation amplified products and the polymorphism in the EST template; and finally, designing a polymorphic EST-SSR site primer to obtain the polymorphic EST-SSR marker. The method is capable of efficiently, simply and conveniently developing the EST-SSR marker and preventing the EST-SSR marker with potential utilizable value from weeding out caused by insufficient genomic sequence or genetic diversity of an experimental material. The developed EST-SSR marker is tightly related to a single gene and has high genetic and breeding values.

Description

technical field [0001] The invention relates to a method for developing polymorphic EST-SSR markers by using whole genome and EST data, and belongs to the field of molecular biology. Background technique [0002] The principle of SSR marking is that the regions on both sides adjacent to the microsatellite sequence are usually highly conserved, and a pair of specific PCR primers can be designed in this conserved region to amplify the microsatellite sequence, and through polyacrylamide gel electrophoresis, The polymorphism of the microsatellite sequence at this locus among individuals can be displayed. Since SSR is randomly distributed in large numbers in the genome, with extensive site variation, it reveals more polymorphisms than RAPD and RFLP, and SSR markers are co-dominant markers, which can distinguish homozygous from heterozygous, Provides complete genetic information, can use PCR method when detecting polymorphism, does not require too many molecular cloning methods, ...

AI Technical Summary

Problems solved by technology

However, when the database is built, the ESTs in the database are obtained by random or shotgun methods by different researchers, which will cause the redundancy of the ESTs.

When developing EST-SSR markers, before searching for SSR sites, the EST data must be compared and spliced ​​to remove redundant sequences. Otherwise, it is very likely to design different primers for the same SSR site, and it will be time-consuming and labor-intensive to cause mis-splicing. possibility, and the removed redundant sequences may contain polymorphisms of SSR length

Combining all current SSR marker methods, newly developed markers usually need to use more than two different genome sequences for polymorphism screening and identification of candidate SSR markers, otherwise it is necessary to use multiple sample DNA with sufficiently large genotype differences for experiments Laboratory screening and verification, during which the genome sequence of the test and SSR markers with no difference between samples will inevitably be eliminated

However, for polymorphic SSR markers with few sources of genome sequence data, small differences, small differences in genotype samples, underrepresentation, and potential utilization value, it is very likely to be eliminated by mistake.

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