Leonardo Congiu

The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application

The random amplified polymorphic DNA (RAPD) technique was applied to settle a lawsuit involving unauthorized commercialization of a patented strawberry variety of high economical relevance (‘Marmolada’®). Because of economical involvements, the molecular approach was added to the more traditional morphological examination in a double‐blind test. All plants belonging to the patented variety were unambiguously identified (13 plants among a total of 31 plants examined). The results were accepted as evidence in the court. This study confirms that the RAPD technique is especially suitable for identification of asexually reproduced plant varieties for forensic or agricultural purposes.

Identification of interspecific hybrids by amplified fragment length polymorphism: the case of sturgeon

The identification of interspecific hybrids represents an important issue for conservation biology and trade controls. In Italy, the commercial demand for sturgeon is rapidly increasing and interspecific hybrids represent a relevant part of aquacultural production. In this study we tested the suitability of the amplified fragment length polymorphism (AFLP) technique for sturgeon hybrid detection. Multilocus AFLP profiles were analysed by cluster analysis and assignment tests based on observed and simulated samples. Our results show that this approach can easily identify sturgeon hybrids, encouraging its application not only in sturgeon but also in other systematic groups.

Nonconcordant evolutionary history of maternal and paternal lineages in Adriatic sturgeon

Although analyses of intraspecific variability are an important prerequisite for species identification assays, only a few studies have focused on population genetics and historical biogeography of sturgeon species. Here we present the first study on genetic variability of the last remaining Adriatic sturgeon, Acipenser naccarii, derived from mitochondrial and nuclear DNA. Our mitochondrial DNA analyses arranged individuals into three distinguished mitochondrial DNA haplogroups (Po1, Po2 and Buna). Two haplogroups (Po1 and Buna) were correlated to geographical distribution, whereas the third (Po2) was not. It was, however, very closely related to one lineage of its Ponto‐Caspian sister species, A. gueldenstaedtii. The distribution of nuclear markers (microsatellites and amplified fragment length polymorphism) was strongly correlated to geographical distribution. An assignment test based on nuclear data placed no specimen of A. naccarii to A. gueldenstaedtii and vice versa. Therefore, the presence of gueldenstaedtii‐like haplotypes within the Po population is either the result of a postglacial introgression or an ancestral polymorphism and does not indicate a hybrid population. The most valuable tool for forensic species identification purposes is one diagnostic deletion separating all A. naccarii from A. gueldenstaedtii. As both A. naccarii populations are genetically differentiated, stocking of sturgeon from the Po River in Italy into waters of the Buna River would jeopardize the genetic differences between both populations and should thus be avoided.

Sturgeon genetics and cytogenetics: recent advancements and perspectives

The aim of this review is to introduce current knowledge in the field of sturgeon genetics. The first section deals with sturgeon cytogenetics, reviewing karyotype organization and polyploidization events during evolution of Acipenseriformes. The second section concerns the results of applications of molecular biology to studies of phylogenetic relationships between extant species, intraspecific analysis of wild populations and stocks for conservation purposes, together with characterization of molecular markers for species identification, relevant to forensic and conservation issues.

The use of bird feathers for the monitoring of cadmium pollution

The cadmium contamination mechanism in bird feathers was investigated using starlings fed with diets containing 10 and 50 ppm Cd for five months. The experiment started about two months before the beginning of the annual complete feather molt and lasted until most of the birds completed the molt of the primaries. Concentrations of Cd in liver, kidney, and uropygial gland were highly correlated, but uropygial gland concentration was about 100 times lower. Cadmium was found both in old and new feathers, in a dose-related manner. Old. feathers showed higher metal concentrations than new ones and primaries higher than secondaries. Feather Cd concentration correlated with Cd concentration in liver, kidney, and the uropygial gland. The use of bird feathers are, therefore, a reliable method for monitoring cadmium pollution, but differences between feather type and age must be considered to correctly interpret data collected in the field.

Sturgeon conservation genomics: SNP discovery and validation using RAD sequencing

Caviar‐producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta‐samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired‐end RAD data focused on the identification of SNPs in the paired‐end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population‐wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping‐by‐sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools.

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.

Molecular cytogenetic analysis of the karyotype of the European Atlantic sturgeon, Acipenser sturio

A karyotype analysis was carried out on the European Atlantic sturgeon, Acipenser sturio (2n=121 ± 3). The telomeric sequence repeat (TTAGGG)n detected by fluorescent in situ hybridization (FISH) was mostly localized at the telomeres of all chromosomes. Ribosomal DNA (rDNA) genes were detected by silver staining techniques and by FISH with digoxigenin-labelled probe for 28S rDNA. Silver staining detected active NORs in the telomeric regions of six chromosomes, and by FISH one or two additional minor sites were detected. The 5S rDNA was found in the interstitial region of a small metacentric pair. The 5S rRNA gene was completely sequenced for the first time in a sturgeon species. The A. sturio karyotype organization is discussed in relation to phylogenesis of the species within the Acipenseridae and to polyploidization events characterizing sturgeon evolution.

Sequencing, De Novo assembly and annotation of the Colorado Potato Beetle, Leptinotarsa decemlineata, Transcriptome.

Background The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest and a serious threat to potato cultivation throughout the northern hemisphere. Despite its high importance for invasion biology, phenology and pest management, little is known about L. decemlineata from a genomic perspective. We subjected European L. decemlineata adult and larval transcriptome samples to 454-FLX massively-parallel DNA sequencing to characterize a basal set of genes from this species. We created a combined assembly of the adult and larval datasets including the publicly available midgut larval Roche 454 reads and provided basic annotation. We were particularly interested in diapause-specific genes and genes involved in pesticide and Bacillus thuringiensis (Bt) resistance. Results Using 454-FLX pyrosequencing, we obtained a total of 898,048 reads which, together with the publicly available 804,056 midgut larval reads, were assembled into 121,912 contigs. We established a repository of genes of interest, with 101 out of the 108 diapause-specific genes described in Drosophila montana; and 621 contigs involved in insecticide resistance, including 221 CYP450, 45 GSTs, 13 catalases, 15 superoxide dismutases, 22 glutathione peroxidases, 194 esterases, 3 ADAM metalloproteases, 10 cadherins and 98 calmodulins. We found 460 putative miRNAs and we predicted a significant number of single nucleotide polymorphisms (29,205) and microsatellite loci (17,284). Conclusions This report of the assembly and annotation of the transcriptome of L. decemlineata offers new insights into diapause-associated and insecticide-resistance-associated genes in this species and provides a foundation for comparative studies with other species of insects. The data will also open new avenues for researchers using L. decemlineata as a model species, and for pest management research. Our results provide the basis for performing future gene expression and functional analysis in L. decemlineata and improve our understanding of the biology of this invasive species at the molecular level.

Phylogenetic relationships of the North-eastern Atlantic and Mediterranean forms of Atherina (Pisces, Atherinidae).

UIE, Instituto Superior de Psicologia Aplicada, Rua Jardim do Tabaco 34, 1149-041 Lisboa, Portugal Departamento de Zoologia e Antropologia, Faculdade de Ciencias da Universidade do Porto, Prac a Gomes Teixeira, 4099-002 Porto, Portugal Dipartamento di Biologia, Universita di Padova, Via U. Bassi 58/B, 35121 Padova, Italy d IMAR/DOP, University of the Azores, Cais Sta Cruz, 9901-862 Horta, Azores, Portugal Centro de Ciencias do Mar do Algarve, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Universidad de La Laguna, Dpto. Biologia Animal (Ciencias Marinas), Av. Astrofisico Francisco Sanchez s/n, 38206 La Laguna, Tenerife, Islas Canarias, Spain g Institute of Fishing Resources, Boulevard Primorski 4, P.O. Box 72, 9000 Varna, Bulgaria h Instituto de Oceanografia, Faculdade de Ciencias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal Department of Biology, Division of Genetics, Cell Biology and Development, University of Patras, Rion-Patras 26500, Greece Museo National de Ciencias Naturales. Jose Gutierrez Abascal 2, 28006 Madrid, Spain