General use molecular marker CNS-AFLP for gramineae

Abstract

The invention discloses a method for building gramineae general molecular marker CNS-AFLP and developed primer sequence with good universality in gramineae crop, which is applicable to the technical fields of genomics, molecular biology and bioinformatics. Coding sequence or EST is taken as a template so as to design the primer; and conserved non-coding sequence (CNS) is taken as the template so as to design the primer. Detecting polymorphism is base sequence length and restriction enzyme cutting site variation of non-coding region between two CNSs and between CNS and Exon. The invention comprises steps of CNS developing, primer designing, PCR amplifying, detecting the polymorphism of amplified fragment, preparing and analyzing the polymorphism of restriction enzyme cutting fragment, etc. Compared with SSR mark, ILP mark and EST mark, the invention is characterized in that transspecies application range is wider and the designed primer is applicable to various species in the gramineae, thereby improving research efficiency. The method is a molecular marking method based on PCR technology, so that the designed primer has high universality and wide transspecies application range, thus effectively promoting the relatively laggard gramineae crop genome research and cereal crop comparative genetics.

Description

technical field [0001] The invention relates to a DNA molecular marker method, which is applied in the three major fields of genomics, molecular biology and bioinformatics, and specifically relates to a method for designing primers based on non-coding conserved sequences (Conserved Noncoding Sequences, CNS) PCR amplification of test species, an analysis method using the polymorphism of the amplified fragment or the enzyme-cleaved product of the amplified fragment as a marker, and the application of this method to develop new general-purpose molecular markers and analysis techniques. Background technique [0002] DNA molecular markers are the basis for gene mapping, cloning, functional studies and marker-assisted selection breeding. Since human geneticist J.G.K.Botstein first proposed the molecular marker technology of DNA restriction fragment length polymorphism in 1980, the first tomato RFLP map came out in 1986, especially after the formation of PCR technology, making DNA ...

AI Technical Summary

Problems solved by technology

The disadvantage of RAPD is that it is generally manifested as dominant inheritance, the information provided is incomplete and in-depth, and it is difficult to distinguish homozygotes and heterozygotes; at the same time, due to the short primers used for RAPD markers, the reaction is easily affected by conditions, and sometimes the band pattern is unstable and repeated. Poor performance, limited application

[0011] The research methods used in the early stages of the comparative genetics of cereals all used the RFLP method based on DNA hybridization. Although it has good versatility among different species of Poaceae, it is a heavy workload, time-consuming, expensive, and polluting. Inconvenience for further in-depth research in the future

Although PCR-based specific markers such as SSR are simple to operate, they have poor versatility in cross-species applications and are difficult to apply in the comparative genetics of different species

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