Molecular marker and detection method for quickly identifying true and false male fish of cynoglossus semilaevis based on RAA-LFD

Abstract

The invention relates to the technical field of molecular biology, and discloses a molecular marker and a detection method for quickly identifying true and false male fish of cynoglossus semilaevis based on RAA-LFD. The method comprises the following steps: S1, selecting a ZW chromosome homologous allele sequence and searching a candidate SNP site; S2, designing a primer pair of an allele sequencecontaining the candidate SNP site by using Primer 3; and S3, carrying out first-generation sequencing on the amplification product of the step S2, and determining a sex specific site through verification of the first-generation sequencing. A new rapid detection method for sex identification is provided, and meanwhile, possibility is provided for on-site timely detection. Generally, laboratory workers or farm farmers only need to set an RAA amplification reaction system and distribute the RAA amplification reaction system into a small centrifuge tube. In the detection process, only sample DNAis added, the product amplification condition can be detected through LFD after incubation is conducted for 40 minutes at the temperature of 39 DEG C, and the detection method has the advantages of being easy and rapid to operate, high in specificity and sensitivity, convenient to detect and the like.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to molecular markers and a detection method for quickly identifying the true and false male sex of half-smooth tongue sole based on RAA-LFD. Background technique [0002] The half-smooth tongue sole belongs to the order Pleuriformes, the family Glossoleidae, and the genus Glossole genus. It is a warm-temperate offshore large-scale bottom fish unique to my country's waters. It is mainly distributed in the coastal areas of the Yellow Sea and the Bohai Sea. Due to the small amount of natural resources, semi-smooth tongue sole, delicious taste, smooth taste and rich nutrition, it has broad breeding prospects and is an important and valuable seawater cultured fish. As a flounder, the growth rate of females of tongue sole is more than three times that of males (Chen et al., 2013), while males have the characteristics of slow growth and small body size, which reduces the culture ...

AI Technical Summary

Problems solved by technology

[0003] In terms of sex-specific molecular marker screening and genetic sex identification of half-smooth tongue sole, previous researchers isolated half-smooth tongue sole female-specific AFLP markers through AFLP (amplified fragment length polymorphism) marker technology. Since AFLP markers are dominantly inherited, they cannot be distinguished in application. ZW females and WW superfemale individuals, it is difficult to avoid false negative results, resulting in misjudgment of ZZ males and ZW females

Through the screening of the co-dominant sex-specific microsatellite scaffold68-2 marker in half-smooth tongue sole, primers can be designed for the co-dominant sex-specific marker loci to identify the genetic sex of half-smooth tongue sole, but in practical applications, the INDEL in the microsatellite is short (less than 50bp), resulting The difference in the fragment lengths of the two PCR amplification products from the Z chromosome and the W chromosome is small, which causes considerable difficulties in determining their lengths by gel electrophoresis

At the same time, in the detection of this technology, agarose gel with a concentration of 4% is needed to help distinguish bands with small length differences (such as less than 50bp), but high-concentration agarose is not easy to prepare, electrophoresis takes a long time, and the cost is high. , which further limits the generalization of the method