Federica Barbisan

Transcriptome sequencing and de novo annotation of the critically endangered Adriatic sturgeon

BackgroundSturgeons are a group of Condrostean fish with very high evolutionary, economical and conservation interest. The eggs of these living fossils represent one of the most high prized foods of animal origin. The intense fishing pressure on wild stocks to harvest caviar has caused in the last decades a dramatic decline of their distribution and abundance leading the International Union for Conservation of Nature to list them as the more endangered group of species. As a direct consequence, world-wide efforts have been made to develop sturgeon aquaculture programmes for caviar production. In this context, the characterization of the genes involved in sex determination could provide relevant information for the selective farming of the more profitable females.ResultsThe 454 sequencing of two cDNA libraries from the gonads and brain of one male and one female full-sib A. naccarii, yielded 182,066 and 167,776 reads respectively, which, after strict quality control, were iterative assembled into more than 55,000 high quality ESTs. The average per-base coverage reached by assembling the two libraries was 4X. The multi-step annotation process resulted in 16% successfully annotated sequences with GO terms. We screened the transcriptome for 32 sex-related genes and highlighted 7 genes that are potentially specifically expressed, 5 in male and 2 in females, at the first life stage at which sex is histologically identifiable. In addition we identified 21,791 putative EST-linked SNPs and 5,295 SSRs.ConclusionsThis study represents the first large massive release of sturgeon transcriptome information that we organized into the public database AnaccariiBase, which is freely available at http://compgen.bio.unipd.it/anaccariibase/. This transcriptomic data represents an important source of information for further studies on sturgeon species. The hundreds of putative EST-linked molecular makers discovered in this study will be invaluable for sturgeon reintroduction and breeding programs.

Managing Polyploidy in Ex Situ Conservation Genetics: The Case of the Critically Endangered Adriatic Sturgeon (Acipenser naccarii)

While the current expansion of conservation genetics enables to address more efficiently the management of threatened species, alternative methods for genetic relatedness data analysis in polyploid species are necessary. Within this framework, we present a standardized and simple protocol specifically designed for polyploid species that can facilitate management of genetic diversity, as exemplified by the ex situ conservation program for the tetraploid Adriatic sturgeon Acipenser naccarii. A critically endangered endemic species of the Adriatic Sea tributaries, its persistence is strictly linked to the ex situ conservation of a single captive broodstock currently decimated to about 25 individuals, which represents the last remaining population of Adriatic sturgeon of certain wild origin. The genetic variability of three F1 broodstocks available as future breeders was estimated based on mitochondrial and microsatellite information and compared with the variability of the parental generation. Genetic data showed that the F1 stocks have only retained part of the genetic variation present in the original stock due to the few parent pairs used as founders. This prompts for the urgent improvement of the current F1 stocks by incorporating new founders that better represent the genetic diversity available. Following parental allocation based on band sharing values, we set up a user-friendly tool for selection of candidate breeders according to relatedness between all possible parent-pairs that secures the use of non-related individuals. The approach developed here could also be applied to other endangered tetraploid sturgeon species overexploited for caviar production, particularly in regions lacking proper infrastructure and/or expertise.

Duplication polymorphism at MHC class II DRB1 locus in the wild boar ( Sus scrofa )

The origin of allelic polymorphism in genes of the major histocompatibility complex represents a central topic in evolutionary genetics as it is probably the most polymorphic region in the nuclear genome of vertebrates. Accordingly, the analyses of genetic variability at these loci provide evidence complementary to the population genetics studies based on neutral loci. In this study, four wild boar populations, two from Italy (Florence region and Castelporziano Presidential Reserve, outside Rome) and one each from Hungary and Poland, were characterized at a highly polymorphic fragment including part of intron 1 and exon 2 of swine leukocyte antigen (SLA) class II DRB1 gene by direct sequencing and by cloning. Excluding the false alleles, a total of 18 different sequences were observed in 57 individuals. The high ratio of nonsynonymous (dN) vs synonymous (dS) substitution rates in the peptide-binding region supports the hypothesis that balancing selection is operating at this locus. A duplication event at the DRB1 gene was documented only in one Italian population with both copies being putatively active. This is the first evidence of a polymorphism for the number of copies of an SLA gene.

Polymorphic microsatellite loci in the widespread amphidromous goby Sicyopterus lagocephalus and cross-genus amplification among Sicydiinae.

Microsatellite DNA markers were isolated in an amphidromous goby (Sicyopterus lagocephalus) from a partial genomic library enriched for AC repeats. Eight microsatellites were highly polymorphic with six to 33 alleles per locus and expected heterozygosities ranging from 0.53 to 0.97. Cross‐species amplifications were performed within the sub‐family Sicydiinae by genotyping individuals from two species of the genus Cotylopus. Some of these loci were successfully amplified and showed polymorphism in the second genus.

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci in the Mediterranean shore crab Carcinus aestuarii (Decapoda, Portunidae)

We characterized nine polymorphic microsatellites in the Mediterranean shore crab Carcinus aestuarii (Decapoda: Portunidae). Microsatellites were isolated from a partial genomic library enriched for multiple motifs. All loci were polymorphic, with number of alleles ranging from two to 16 and a mean observed heterozygosity of 0.75. Seven loci were in Hardy–Weinberg equilibrium, and two showed weak heterozygote deficiency. No linkage disequilibrium was found between loci. In addition, we tested Hardy–Weinberg and linkage equilibrium of three Carcinus maenas loci, already reported to cross‐amplify in C. aestuarii. These molecular markers will be potentially useful to investigate genetic structure of this species.

Isolation, characterization and cross‐species testing of microsatellites obtained from a sand smelt (Atherina boyeri) genomic library

This study reports the isolation and characterization of 11 polymorphic microsatellites from a sand smelt (Atherina boyeri) genomic library. Enrichment was performed with di‐, tri‐ and tetranucleotide motifs following the FIASCO procedure (fast isolation by AFLP of sequences containing repeats). All loci were found to be in linkage and in Hardy–Weinberg equilibrium. This represents the first microsatellite isolation for the family Atherinidae and the isolated loci were accordingly tested on four additional species of the family: two recognized (A. presbyter and A. hepsetus) and two proposed (‘punctata’ and ‘non‐punctata’ forms). Moreover their cross‐species suitability on Menidia menidia, belonging to the same order but to the family Atherinopsidae, was also tested.