Spatiotemporal expression of zebrafish D-amino acid oxidase during early embryogenesis

[[abstract]]D-amino acid oxidase (DAO, EC 1.4.3.3) is one of the flavoenzymes in peroxisomes catalyzing the oxidation of D-amino acids to α-imino acids. By reverse transcription-polymerase chain reaction, DAO cDNA was cloned from the mRNA of zebrafish embryos. The deduced zebrafish DAO amino acid sequence revealed a 348-amino acid polypeptide containing two FAD-binding sites (amino acids positions 7G to 12G, and 236V to 247W), and a peroxisomal targeting signal sequence (346Ser-347Arg-348Leu) at the C-terminal end. Following comparison of the sequences, we found that the zebrafish DAO polypeptide shared the sequence identities of 93%, 79%, 77%, 77%, 46%, 43% and 25% of the reported DAO of carp, human, mouse, pig, Streptomyces coelicor, Rhodotorula gracilis and Schizosaccharomyces pombe, respectively. Whole-mount in situ hybridization revealed that dao was a maternally inherited gene and that its expression was first observed in the zygote period, gradually down-regulated from the cleavage period to the early blastula stages, and had declined to an undetectable level at 6 h post-fertilization (hpf). At 18-hpf, the zebrafish dao transcripts were detected in a very weak manner, and their expression was restricted in the future head and brain regions as well as in the yolk syncytial layers of the yolk and yolk extension regions. At later stages, dao expression was detected in the brain, gut, swimming bladder, pronephric duct, optic tectum, ventrolateral tract and in the floor plate of the developing spinal cord. The enzymatic activities of DAO were also examined and showed that the DAO enzyme activities were expressed in a dynamic and stage-dependent manner: first it was detected at the 1-cell stage at an extremely low level (0.007 ± 0.005 U/mg), reached the maximum amount at 6 days post-fertilization (dpf) (0.323 ± 0.04 U/mg), and gradually down-regulated to the basal level (0.019 ± 0.005 U/mg) in a 1-month juvenile. From these observations, we conclude that zebrafish dao is a maternally inherited gene, and that its expression was restricted in some neurons, gut, and pronephros and could be used as a good marker for the study of pronephridial development.[[notice]]補正完畢[[journaltype]]國外[[incitationindex]]SCI[[booktype]]紙本[[booktype]]電子版[[countrycodes]]NL

Female-specific SNP markers provide insights into a WZ/ZZ sex determination system for mud crabs Scylla paramamosain, S. tranquebarica and S. serrata with a rapid method for genetic sex identification

Abstract Background Mud 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. Results Restriction-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. Conclusions Sex-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