Sex-specific DNA Molecular Makers In Chinese Giant Salamander And Analysis Of Differentially Expressed Genes Between Female And Male Gonad

The Chinese giant salamander is peculiar endangered caudata amphibians in China. It has important function to study the origin, evolution, genetic diversity and sex determination of amphibian because of its special evolutionary history. Recently, because of the habitat destruction and capturing and their low fertility, the number of the wild Chinese Giant Salamander is smaller and smaller. Although many places are carrying out the artificial breeding, the problem of low fertility has still not been solved because of its hard to distinguish between male and female. In order to improve the breeding efficiency and protect the wild species, we want to find sex-specific DNA marker to distinguish the female and the male and we try to explore the expression of the main genes related to gonad development by transcriptome sequencing.The main results of this paper are described as follows:(1) We collected 40 female and 40 male Chinese Giant Salamander muscle DNA. We diluted every DNA sample into 50ng/ul and then mix the DNA of the same gender as the same amount, forming two DNA pool. Then we synthesize the 100 RAPD primers and amplified the templates by PCR in the DNA pools. As a result, we got 6 sex-specific RAPD primers. However, we found that primer s200 can separated the female and the male in different individuals in 24 DNA samples(12 female and 12male).(2) We sequenced the female-specific band by RAPD s200 primer and got one fragments of 1270 bp fragment. Then we design 4 pairs of primers to amplify the DNA pool, finding that only primer F96 and R477 can distinguish the female and the male. We test the F96 and R477 primers by 20 female individuals and 20 male individuals, showing that the primers can distinguish the gender in all the samples. In addition, we can also replicate the result in the ecdysis. We amplify the female and male individuals by primer F96 and R1259 and then sequence the product. We find many mutations between the female and the male and there are three SNP in the location of R477 primer. What’s more, we got 1000 bp fragment in the 3’ end by genomic walking and we also find many mutation in the 1000 bp fragment, which will help us find more sex-specific DNA markers.(3) Male and female Chinese Giant Salamander are difficult to distinguish just based on the shape. The gonads of four-year-old the Chinese Giant Salamander are got and we did histology observation of testis and ovary, including the hematoxylin-eosin staining. It show obvious difference and it probably be related to the differences of the gene expression. Transcriptome sequencing was carried on and we find a lot of differentially expressed genes by bioinformatics analysis. Then we predict 4 Unigene can involve in the process of sex determination and 5 Unigene can play an important role in the development of gonad by GO analysis. We also find 15 sex determination genes in other species, which have a high similarity to some fragments in the contig data. We can predict that these genes may have similar function in the Chinese Giant Salamander.