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Draft Genome and Complete Hox-Cluster Characterization of the Sterlet (Acipenser ruthenus).

ABSTRACT: Background: Sturgeons (Chondrostei: Acipenseridae) are a group of "living fossil" fishes at a basal position among Actinopteri. They have raised great public interest due to their special evolutionary position, species conservation challenges, as well as their highly-prized eggs (caviar). The sterlet, Acipenser ruthenus, is a relatively small-sized member of sturgeons and has been widely distributing in both Europe and Asia. In this study, we performed whole genome sequencing, de novo assembly and gene annotation of the tarlet to construct its draft genome. Findings: We finally obtained a 1.83-Gb genome assembly (BUSCO completeness of 81.6%) from a total of 316.8-Gb raw reads generated by an Illumina Hiseq 2500 platform. The scaffold N50 and contig N50 values reached 191.06 and 18.88 kb, respectively. The sterlet genome was predicted to be comprised of 42.84% repeated sequences and to contain 22,184 protein-coding genes, of which 21,112 (95.17%) have been functionally annotated with at least one hit in public databases. A genetic phylogeny demonstrated that the sterlet is situated in the basal position among ray-finned fishes and 4dTv analysis estimated that a recent whole genome duplication occurred 21.3 million years ago. Moreover, seven Hox clusters carrying 68 Hox genes were characterized in the sterlet. Phylogeny of HoxA clusters in the sterlet and American paddlefish divided these sturgeons into two groups, confirming the independence of each lineage-specific genome duplication in Acipenseridae and Polyodontidae. Conclusions: This draft genome makes up for the lack of genomic and molecular data of the sterlet and its Hox clusters. It also provides a genetic basis for further investigation of lineage-specific genome duplication and the early evolution of ray-finned fishes.

SUBMITTER: Cheng P

PROVIDER: S-EPMC6739705 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.

Project description:Sturgeons, producers of black caviar, are one of the most valuable wildlife commodities on earth (Pikitch et al., 2005). They appeared approximately 200 million years ago, and are known as living fossils (Bemis et al., 1997). Sturgeon populations dramatically declined as a result of overfishing, poaching and habitat destruction (Birstein, 1993; Billard and Lecointre, 2001; Vidotto et al., 2013). International Union for Conservation of Nature and Natural Resources (IUCN) classified over 85% of sturgeon species as at risk of extinction, more than any other group of species. Therefore, artificial reproduction techniques have been developed for sturgeons to meet the demands of aquaculture and restocking programs. Basic knowledge regarding biology of reproduction such as egg activation and fertilization can help improving the efficiency of artificial reproduction. Fertilization happens when gametes, spermatozoon and egg, are fused together. Egg activation refers to a series of morphological and molecular changes that occur in the egg during and after fertilization. Egg activation is believed to prevent polyspermy, and may also establish a micro-environment to support embryo development (Wong and Wessel, 2006; Niksirat et al., 2015a). Although, earlier researchers have illustrated egg structure and its morphological changes during egg fertilization and activation in sturgeons (Cherr and Clark, 1982, 1985a; Linhart and Kudo, 1997; Debus et al., 2008; Zelazowska, 2010), there is still a lack of molecular knowledge to improve our understanding of this stage of reproduction in these animals. Eggs of sturgeon are released to aqueous environment, where they are fertilized, activated and develop an adhesive layer after contact with freshwater (Cherr and Clark, 1984; Vorobeva and Markov, 1999). Although, egg stickiness is necessary for eggs in nature to help them find a stable position by attaching to substrates such as gravel and stone and increase chance of survival until hatching (Riehl and Patzner, 1998), but can cause high rate of mortality during artificial incubation as a result of attachment of eggs together and subsequent suffocation in limited space of incubators. Using clay suspension in water as an activation medium has been proven to be effective to block egg stickiness in sturgeon eggs (Dettlaff et al., 1993). In recent years, label-free protein quantification techniques have been developed based on the presumption of linear proportionality between peptide mass peak signal intensity and concentration of a given peptide. This approach avoids the limitations and multiple preparation steps of methods such as those based on two-dimensional gel electrophoresis (Niksirat et al., 2015b). The aim of present study was to identify and quantify proteome changes of sterlet eggs during activation in different activation media, water and clay suspension, using label-free protein quantification technique.

Project description:In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.

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Draft Genome and Complete Hox-Cluster Characterization of the Sterlet (Acipenser ruthenus).

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