Method for screening Andrias davidianus female specific fragment and detecting genetic sexuality through RAD sequencing

Abstract

The invention discloses a method for screening Andrias davidianus female specific fragment and detecting genetic sexuality through RAD sequencing. The method comprises: 1) scanning of Andrias davidianus male-female genome pool through RAD sequencing technology and data analysis: carrying out genomic DNA extraction, enzymatic digestion, library construction and sequencing to obtain Andrias davidianus male-female genome RAD data, and analyzing the Andrias davidianus male-female genome RAD data to obtain male-female specific candidate RAD data; 2) verification of candidate female specific fragment: selecting female specific candidate fragments, designing primers, and carrying out PCR amplification by using DNA as a template, wherein the characteristics that the fragment with the expected sizecan be amplified, and the specific band only exists in the female individual and does not exist in the male individual are used as the specific marker; and 3) sequence analysis of the female specificfragment: cloning the specific fragment into a PMD-18T vector, ligating, transfecting, detecting positive clones by using a conventional method, sending samples, and sequencing, wherein the homologyreached 96%. According to the present invention, the method has advantages of accuracy, easy performing, low cost and simple operation, and can solve the problem of Andrias davidianus gender identification in the current production.

Description

technical field [0001] The invention belongs to the technical field of Chinese giant salamander molecular markers and genetic sex identification, and more specifically relates to a RAD sequencing method for screening Chinese giant salamander female-specific fragments and a genetic sex detection method, which is suitable for genetic sex identification applied in giant salamander breeding and sex-controlled breeding. Background technique [0002] Chinese giant salamander ( Andrias davidianus ), also known as "Salamosaurus", is the largest freshwater amphibian in existence in the world. In the 1980s, the wild giant salamander was listed as a second-class national key protected tailed amphibian (Ye Changyuan et al., 1993). In addition, the Chinese giant salamander is also an economic animal with high edible and medicinal value. It has a unique flavor and has the effects of nourishing yin and kidney, nourishing blood and promoting qi (Zhang Shenhu, 2001), known as "ginseng in wa...

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Problems solved by technology

The accuracy of the above methods is not high or the actual operation is difficult and expensive

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