High-efficiency screening method for microsatellite marker of Marbled Sand Goby and application of same

Abstract

The invention belongs to the technical field of DNA markers of molecular genetics of fishes, and particularly relates to the fast and highly efficient screening and application of the microsatellite loci of the Marbled Sand Goby. The method comprises the following steps: large mass DNA enzyme ligation, connection; low cycle index Pre-PCR; construction of the enriched library of microsatellite fragments by utilizing a biotinylated probe and streptomycin magnetic beads; screening of positive clones, and further improvement of the detection efficiency of the microsatellite loci through secondary screening; and population genetic analysis by using an obtained microsatellite primer. The invention improves the existing magnetic-bead enrichment method, efficiently and quickly screens the microsatellite loci, reduces the sequencing cost, and simultaneously overcomes the deficiency on safety during secondary screening adopting isotope. The obtained microsatellite marker provides scientific basis and detection means for the germ plasm resource protection, the germ plasm improvement, the genetic breeding, and the like of the Marbled Sand Goby.

Description

technical field [0001] The invention belongs to the technical field of fish molecular genetics DNA markers, and in particular relates to rapid and efficient screening and application of microsatellite sites (SSR) of Oxyeleotris armoratus. Background technique [0002] Commonly known as Marble goby, it belongs to the order Pereiformes, the family Eleotridae, and the genus Oxyeleotris. Snakehead is a large and rare fish of the family Snakenaceae native to Southeast Asian countries such as Thailand and Laos. It is very popular among consumers in coastal cities of my country, Hong Kong, Macao and Taiwan, and has high economic value. Since my country introduced from Southeast Asia, it has become an important freshwater aquaculture object in the southern coastal areas of my country. [0003] Due to small population breeding, inbreeding and non-random mating factors in the process of artificial breeding, problems such as reduced genetic variation level, degraded traits and reduced...

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Problems solved by technology

However, there are also some problems in the traditional library enrichment method: 1. In order to offset the loss of the DNA fragments produced by the enrichment step and the conversion step, the library fragments are generally increased in a large amount by adding two PCR steps of 25-35 cycles, so that It will lead to a substantial increase in the number of certain DNA fragments to become dominant fragments, resulting in the same colony clones, and finally the number of microsatellite sites screened each time is still relatively limited; 2. Although the proportion of microsatellites screened is higher than that of direct screening methods, However, the final sequencing results show that only 30%-40% of the enriched fragments still contain microsatellite sites; 3. The method of isotope probe hybridization can identify whether microsatellite sites are included in the colony, and improve the efficiency of screening, but This method has complicated steps, and general laboratories do not have the conditions to carry out isotope work, thus limiting the scope of application

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