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Abstract
BackgroundMud crabs, Scylla spp., are commercially important large-size marine crustaceans in the Indo-West Pacific region. As females have the higher growth rate and economic value, the production of all female stocks is extremely essential in aquaculture. However, the sex determination mechanism is still unclear. Development of sex-specific genetic markers based on next-generation sequencing proved to be an effective tool for discovering sex determination system in various animals.ResultsRestriction-site associated DNA sequencing (RAD-seq) was employed to isolate sex-specific SNP markers for S. paramamosain. A total of 335.6 million raw reads were obtained from 20 individuals, of which 204.7 million were from 10 females and 130.9 million from 10 males. After sequence assembly and female-male comparison, 20 SNP markers were identified to be sex-specific. Furthermore, ten SNPs in a short sequence (285 bp) were confirmed heterozygous in females and homozygous in males in a large population by PCR amplification and sequencing. Subsequently, a female-specific primer was successfully designed according to the female-specific nucleotide which could amplify an expected band from females but not from males. Thus, a rapid and effective method for molecular sexing in S. paramamosain was developed, meanwhile, this method could successfully identify the sex of S. tranquebarica and S. serrata. Finally, nine and four female-specific SNP markers were detected in S. tranquebarica and S. serrata, respectively.ConclusionsSex-specific SNP markers were firstly identified in crab species and showed female heterogamety and male homogamety, which provided strong genetic evidence for a WZ/ZZ sex determination system in mud crabs S. paramamosain, S. tranquebarica and S. serrata. These findings will lay a solid foundation for the study of sex determination mechanism, sex chromosome evolution, and the development of mono-sex population in crustaceans.
Highlights
Mud crabs, Scylla spp., are commercially important large-size marine crustaceans in the Indo-West Pacific region
RAD sequencing A total of 335,600,178 raw reads were generated after Restriction-site associated DNA sequencing (RAD-seq), with 204,685,780 for females and 130,914,398
To the best of our knowledge, this is the first study to identify female-specific single nucleotide polymorphisms (SNPs) markers in mud crab (S. paramamosain) that showed heterozygous in females but homozygous in males
Summary
Scylla spp., are commercially important large-size marine crustaceans in the Indo-West Pacific region. The sex determination mechanism is still unclear. Development of sexspecific genetic markers based on next-generation sequencing proved to be an effective tool for discovering sex determination system in various animals. Several crab species, including Plagusia dentipes, Eriocheir japonicus and Hemigrapsus sanguineus, are thought to have a XY/ XX sex determination system based on karyotype studies [6–8]. A recent study suggested that Chinese mitten crab (Eriocheir sinensis) exhibited a WZ/ZZ sex determination system according to quantitative trait locus location of the gender phenotype [5]. Studies on the genetic basis of sex determination mechanism are the foundation towards future sex manipulation biotechnologies, including the development of mono-sex population [2, 12], especially for those crabs with significant sexual dimorphism, such as caribbean king crab (Mithrax spinosissimus) [13] and Japanese mitten crab (Eriocheir japonica) [14]. The sex determination mechanism remains unclear in most aquaculture crustacean species, which have obviously limited its potential application in the aquaculture sector
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