Abstract
BackgroundChinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism.ResultsWe used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17β-estradiol exposed group.ConclusionThis is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation.
Highlights
Chinese giant salamander Andrias davidianus is an endangered species
The ligation was performed via polymerase chain reaction (PCR) at 37 °C for 30 min followed by 65 °C for 30 min
Restriction site-associated DNA sequencing and assembling The restriction-site-associated DNA (RAD)-Seq of four male and four female A. davidianus was performed on an Illumina HiSeq PE150 sequencing platform, and 93.4 million reads and 134,377,511,348 bp of data were generated
Summary
The success of artificial breeding provides a useful way to protect this species. The method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism. Several techniques can be employed to identify chromosomal sex. The karyotype is visualized by cytogenetic techniques to detect the heteromorphic sex chromosome [5,6,7]. Breeding of sex reversed neomales and neofemales can reveal which sex is heteromorphic based on the sex ratio of the progeny [8,9,10,11,12].
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