Analisi di 3 microdelezioni implicate nel disturbo autistico

Nella Tesi viene riportata l’analisi genetica di un campione di 128 famiglie con Disturbo dello Spettro Autistico, tramite il sistema di SNP array “PsychArray” (Illumina ), contenente oltre 500.000 sonde sull’intero genoma. Questi dati sono stati utilizzati per individuare Copy Number Variants (CNVs) rari e rilevanti da un punto di vista clinico. Sono stati quindi selezionati tre CNVs per un ulteriore approfondimento: due microdelezioni già descritte come patologiche (rispettivamente nella regione 1p36.32 e 22q13.33 comprendente il gene SHANK3) sono risultate essere “de novo”, mentre una terza microdelezione nel gene CTNNA3 è ereditata dalla madre. Tutti e tre i CNV sono stati validati tramite Real Time-PCR, definendone i confini. Per quanto riguarda la microdelezione in CTNNA3, poiché difetti di questo gene sono stati implicati nell’autismo con un meccanismo recessivo, è stata anche condotta un’analisi di sequenza di tutti gli esoni del gene negli individui della famiglia interessata, al fine di ricercare eventuali mutazioni puntiformi sull’allele non deleto. Questa analisi non ha individuato nessuna variante potenzialmente dannosa, pertanto il difetto in CTNNA3 non risulta essere la causa principale del fenotipo autistico in questa famiglia, anche se potrebbe avere un ruolo come fattore di suscettibilità.

Identification of stock units of Mullus barbatus (Linnaeus, 1758) in the Italian seas: development of genomic 2b-RAD markers and biological variation analysis.

This thesis is settled within the STOCKMAPPING project, which represents one of the studies that were developed in the framework of RITMARE Flagship project. The main goals of STOCKMAPPING were the creation of a genomic mapping for stocks of demersal target species and the assembling of a database of population genomic, in order to identify stocks and stocks boundaries. The thesis focuses on three main objectives representing the core for the initial assessment of the methodologies and structure that would be applied to the entire STOCKMAPPING project: individuation of an analytical design to identify and locate stocks and stocks boundaries of Mullus barbatus, application of a multidisciplinary approach to validate biological methods and an initial assessment and improvement for the genotyping by sequencing technique utilized (2b-RAD). The first step is the individuation of an analytical design that has to take in to account the biological characteristics of red mullet and being representative for STOCKMAPPING commitments. In this framework a reduction and selection steps was needed due to budget reduction. Sampling areas were ranked according the individuation of four priorities. To guarantee a multidisciplinary approach the biological data associated to the collected samples were used to investigate differences between sampling areas and GSAs. Genomic techniques were applied to red mullet for the first time so an initial assessment of molecular protocols for DNA extraction and 2b-RAD processing were needed. At the end 192 good quality DNAs have been extracted and eight samples have been processed with 2b-RAD. Utilizing the software Stacks for sequences analyses a great number of SNPs markers among the eight samples have been identified. Several tests have been performed changing the main parameter of the Stacks pipeline in order to identify the most explicative and functional sets of parameters.

Stock boundaries of Parapenaeus longirostris (Lucas, 1846) populations in the Italian seas: Development of genomic 2b-RAD markers and biological variation analysis.

This thesis is developed in the contest of Ritmare project WP1, which main objective is the development of a sustainable fishery through the identification of populations boundaries in commercially important species in Italian Seas. Three main objectives are discussed in order to help reach the main purpose of identification of stock boundaries in Parapenaeus longirostris: 1 -Development of a representative sampling design for Italian seas; 2 -Evaluation of 2b-RAD protocol; 3 -Investigation of populations through biological data analysis. First of all we defined and accomplished a sampling design which properly represents all Italian seas. Then we used information and data about nursery areas distribution, abundance of populations and importance of P. longirostris in local fishery, to develop an experimental design that prioritize the most important areas to maximize the results with actual project funds. We introduced for the first time the use of 2b-RAD on this species, a genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Thanks to this method we were able to move from genetics to the more complex genomics. In order to proceed with 2b-RAD we performed several tests to identify the best DNA extraction kit and protocol and finally we were able to extract 192 high quality DNA extracts ready to be processed. We tested 2b-RAD with five samples and after high-throughput sequencing of libraries we used the software “Stacks” to analyze the sequences. We obtained positive results identifying a great number of SNP markers among the five samples. To guarantee a multidisciplinary approach we used the biological data associated to the collected samples to investigate differences between geographical samples. Such approach assures continuity with other project, for instance STOCKMED, which utilize a combination of molecular and biological analysis as well.

Temporal variation of genetic diversity and differentiation of albacore, Thunnus alalunga, in the Mediterranean Sea

This study is on albacore (Thunnus alalunga, Bonnaterre 1788), an epi- and mesopelagic oceanic tuna species cosmopolitan in the tropical and temperate waters of all oceans including the Mediterranean Sea, extending in a broad band between 40°N and 40°S. What it’s known about albacore population structure is based on different studies that used fisheries data, RFLP, mtDNA control region and nuDNA markers, blood lectins analysis, individual tags and microsatellite. At the moment, for T. alalunga six management units are recognized: the North Pacific, South Pacific, Indian, North Atlantic, South Atlantic and Mediterranean stocks. In this study I have done a temporal and spatial comparison of genetic variability between different Mediterranean populations of Thunnus alalunga matching an historical dataset ca. from 1920s composed of 43 individuals divided in 3 populations (NADR, SPAIN and CMED) with a modern dataset composed of 254 individuals and 7 populations (BAL, CYP, LIG, TYR, TUR, ADR, ALB). The investigation was possible using a panel of 94 nuclear SNPs, built specifically for the target species at the University of Basque Country UPV/EHU. First analysis done was the Hardy-Weinberg, then the number of clusters (K) was determined using STRUCTURE and to assess the genetic variability, allele frequencies, the average number of alleles per locus, expected (He) and observed (Ho) heterozygosis, and the index of polymorphism (P) was used the software Genetix. Historical and modern samples gives different results, showing a clear loss of genetic diversity over time leading to a single cluster in modern albacore instead of the two found in historical samples. What this study reveals is very important for conservation concerns, and additional research endeavours are needed.

Are there genetic breaks between Atlantic and Pacific Yellowfin tuna (Thunnus albacares) populations? A preliminary study based on microsatellites gene variation

Yellowfin tuna (Thunnus albacares, YFT, Bonnaterre 1788) is one of the most important market tuna species in the world. The high mortality of juveniles is in part caused by their bycatch. Indeed, if unregulated, it could permanently destabilize stocks health. For this reason investigating and better knowing the stock boundaries represent a crucial concern. Aim of this thesis was to preliminary investigate the YFT population structure within and between Atlantic and Pacific Oceans through the analysis of genetic variation at eight microsatellite loci and assess the occurrence of barriers to the gene flow between Oceans. For this propouse we collected 4 geographical samples coming from Atlantic and Pacific Ocean and selected a panel of 8 microsatellites loci developped by Antoni et al., (2014). Samples 71-2-Y and 77-2-Y, came from rispectively west central pacific ocean (WCPO) and east central pacific ocean (ECPO), instead samples 41-1-Y and 34-2-Y derive from west central atlantic ocean (WCAO) and east central atlantic ocean (ECAO). Total 160 specimens were analyzed (40 per sample) and were carried out several genetic information as allele frequencies, allele number, allelic richness, HWE (using He and Ho) and pairwise Fst genetic distance. Results obtained, may support the panmictic theory of this species, only one of pairwise Fst obtained is statistically significant (Fst= 0.00927; pV= 0.00218) between 41-1-Y and 71-2-Y samples. Results suggest low genetic differentiation and consequent high level of gene flow between Atlantic and Pacific populations. Furthermore, we performed an analysis of molecular taxonomy through the use of ATCO (the flaking region between ATPse6 and cytochrome oxidase subunit III genes mt DNA, to discriminate within the gener Thunnus two of the related species (Yellofin and bigeye tuna) according with their difficult recognition at certain size (<40 cm). ATCO analysis in this thesis, has provided strong discriminate evidence between the target species proving to be one of the most reliable genetic tools capable to indagate within the genus Thunnus. Thus, our study has provided useful information for possible use of this protocol for conservation plans and management of this fish stocks.

First assessment on genetic structure and phylogeography of Mediterranean blue shark (prionace glauca, L. 1758) population using mitochondrial gene variation: a comparison with the Atlantic.

The blue shark, Prionace glauca, is one of the most vagile shark species worldwide distributed. The particular body shape allows blue sharks make transoceanic movements, leading to a circumglobal distribution. Due to its reproductive cycle, an extraordinarily high number of specimens is globally registered but, even if it is still a major bycatch of longline fishery rather than a commercial target, it is characterized by a high vulnerability. In this perspective it is important to increase the amount of informations regarding its population extent in the different worldwide areas, evaluating the possible phylogeographic patterns between different locations. This study, included in the "MedBlueSGen" European project, aims exactly at filling a gap in knowledges regarding the genetic population structure of the Mediterranean blue sharks, which has never been investigated before, with a comparison with the North-Eastern Atlantic blue shark population. To reach this objective, we used a dataset of samples from different Mediterranean areas implementing it with some samples from North-Eastern Atlantic. Analyzing the variability of the two mitochondrial markers control region and cytochrome b, with the design of new species-specific primer pairs, we assessed the mitochondrial genetic structure of Mediterranean and North-Eastern Atlantic samples, focusing on the analysis of their possible connectivity, and we tried to reconstruct their demographic history and population size. Data analyses highlighted the absence of a genetic structuring within the Mediterranean and among it and North-Eastern Atlantic, suggesting that the Strait of Gibraltar doesn't represent a phylogeographic barrier. These results are coherent to what has been found in similar investigations on other worldwide blue shark populations. Analysis of the historical demographic trend revealed a general stable pattern for the cytochrome-b and a slightly population expansion for the control region marker.