GENETIC AND GENOMIC INVESTIGATIONS FOR THE CONSERVATION OF STURGEONS

The order of Acipenseriformes is one of the most endangered taxa. One paddlefish species and another sturgeon species have been recently declared respectively ‘Extinct’ and ‘Extinct in the Wild’ according to the IUCN Red List’s assessment. Of the remnant twenty-five species, twenty are classified either as ‘Endangered’ or ‘Critically Endangered’ while the last five are stated as ‘Vulnerable’. Therefore, these populations require timely and adequate conservation plans both in situ and ex situ that need to be informed by relatedness analysis and genetic variability characterization. The present Ph.D. thesis will mainly focus on the Adriatic sturgeons (Acipenser naccarii), a tetraploid species endemic to the Adriatic region, and historically present in the Po Rivers and its tributaries. Intending to preserve the highest possible level of genetic variability in future generations the farmed progenies (F1), now mature, need to be allocated to their parents of wild origin (F0) to identify groups of sibs and to inform breeding plans. To this purpose, microsatellite loci will be used as they still be the best markers to study polyploid species. However, before applying microsatellite markers to parental allocations, the segregation patterns at these loci need to be previously assessed. In fact, different possible inheritance patterns are possible in tetraploids, namely disomic and tetrasomic. A similar microsatellite-based relatedness analysis was then applied to another sturgeon species on the brink of extinction, the Chinese sturgeon (Acipenser sinensis), to estimate the minimum breeders’ number that participated in the only significant breeding event occurs in the last 10. Even in this case microsatellite loci, resulted to be powerful molecular markers for parental inferences, allowing to estimate an approximative number of 10 males and 10 females remaining for this species. The Adriatic sturgeon was also included in a PRIN project called ENDEMIXIT (https://endemixit.com/) whose aim is to apply conservation genomics to five endangered Italian endemics. Given the possibility to perform controlled reproductions, this species was selected to test in vivo the potentiality and weakness of genomic mutation load predictions, evaluating the relations between the load of bioinformatically identified deleterious mutations and the offspring fitness of selected parents. Another relevant contribution of genetics to the management of captive stocks is the development of suitable markers for the identification of interspecific hybrids which are frequently occurring in captive stocks due to the promiscuity between aquaculture for commercial production and aquaculture for conservation. To develop a new category of multilocus markers (MIPs, Multi-locus Intron Polymorphisms) that allow the detection of multi-species contribution also in hybrids after the first generation, the possibility to use the polymorphisms of intronic regions was explored. This study followed a more general investigation on Teleosts in which thanks to the numerous available genomes, a highly transferable panel of primers for intronic regions amplification was identified and a protocol for simultaneous amplification followed by sequencing on Illumina platform was optimized. Introns proved to be promising markers at both species and population levels in different teleost species. For this reason, the same approach was applied to sturgeons. MIPs markers designed for sturgeons promise to be highly versatile with many possible applications from individuals to populations levels in detecting differentiation signals. An important tool for the analyses of genetic diversity in situ and ex situ conservation plans, and the identification of species and hybrids to contrast the illegal trade of surgeons’ derived products.The order of Acipenseriformes is one of the most endangered taxa. One paddlefish species and another sturgeon species have been recently declared respectively ‘Extinct’ and ‘Extinct in the Wild’ according to the IUCN Red List’s assessment. Of the remnant twenty-five species, twenty are classified either as ‘Endangered’ or ‘Critically Endangered’ while the last five are stated as ‘Vulnerable’. Therefore, these populations require timely and adequate conservation plans both in situ and ex situ that need to be informed by relatedness analysis and genetic variability characterization. The present Ph.D. thesis will mainly focus on the Adriatic sturgeons (Acipenser naccarii), a tetraploid species endemic to the Adriatic region, and historically present in the Po Rivers and its tributaries. Intending to preserve the highest possible level of genetic variability in future generations the farmed progenies (F1), now mature, need to be allocated to their parents of wild origin (F0) to identify groups of sibs and to inform breeding plans. To this purpose, microsatellite loci will be used as they still be the best markers to study polyploid species. However, before applying microsatellite markers to parental allocations, the segregation patterns at these loci need to be previously assessed. In fact, different possible inheritance patterns are possible in tetraploids, namely disomic and tetrasomic. A similar microsatellite-based relatedness analysis was then applied to another sturgeon species on the brink of extinction, the Chinese sturgeon (Acipenser sinensis), to estimate the minimum breeders’ number that participated in the only significant breeding event occurs in the last 10. Even in this case microsatellite loci, resulted to be powerful molecular markers for parental inferences, allowing to estimate an approximative number of 10 males and 10 females remaining for this species. The Adriatic sturgeon was also included in a PRIN project called ENDEMIXIT (https://endemixit.com/) whose aim is to apply conservation genomics to five endangered Italian endemics. Given the possibility to perform controlled reproductions, this species was selected to test in vivo the potentiality and weakness of genomic mutation load predictions, evaluating the relations between the load of bioinformatically identified deleterious mutations and the offspring fitness of selected parents. Another relevant contribution of genetics to the management of captive stocks is the development of suitable markers for the identification of interspecific hybrids which are frequently occurring in captive stocks due to the promiscuity between aquaculture for commercial production and aquaculture for conservation. To develop a new category of multilocus markers (MIPs, Multi-locus Intron Polymorphisms) that allow the detection of multi-species contribution also in hybrids after the first generation, the possibility to use the polymorphisms of intronic regions was explored. This study followed a more general investigation on Teleosts in which thanks to the numerous available genomes, a highly transferable panel of primers for intronic regions amplification was identified and a protocol for simultaneous amplification followed by sequencing on Illumina platform was optimized. Introns proved to be promising markers at both species and population levels in different teleost species. For this reason, the same approach was applied to sturgeons. MIPs markers designed for sturgeons promise to be highly versatile with many possible applications from individuals to populations levels in detecting differentiation signals. An important tool for the analyses of genetic diversity in situ and ex situ conservation plans, and the identification of species and hybrids to contrast the illegal trade of surgeons’ derived products

Characterization of Captive Breeders to Preserve the Residual Genetic Diversity of Adriatic Sturgeon (Acipenser naccarii)

Since 1996, the Adriatic sturgeon (Acipenser naccarii) has been inscribed on the IUCN Red List as "Critically Endangered and possibly extinct in the wild". Nowadays, its survival totally depends on restocking programs conducted by releasing juveniles generated from adult breeders reared in aquaculture. Conducting accurate genetic characterizations of all individuals potentially involved in reproduction activities is therefore of primary importance to avoid inbreeding and to maximize the genetic diversity transmitted to following generations. Since all animals reared in captivity descend from a single stock of wild origin, this offers the ideal condition for carrying out relatedness analysis based on parentage allocations. In this study, we provided the most complete characterization of about 500 individuals representing the most diverse extant stock of Adriatic sturgeon. Through the analyses of mitochondrial d-loop and 15 microsatellite loci selected from 24 genotyped loci, we identified about 30 different familiar groups, updating data on breeding stocks, increasing the genetic information already available, and extending the analyses to animals never genotyped before. Given its completeness, it will represent a reference database for any future parental allocation of recaptured animals for the inclusion of all other stocks present, as well as for the development of a long-term breeding plan. The approach used has also been proven useful on individuals of unknown genealogy, allowing for the identification of family groups and thus being proven to be promising for the analysis of stocks of other tetraploid sturgeon species

Different Chromosome Segregation Patterns Coexist in the Tetraploid Adriatic Sturgeon Acipenser naccarii

The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a critically endangered tetraploid endemism of the Adriatic region; it has been targeted, over the last 20 years, by different conservation programs based on controlled reproduction of captive breeders followed by the release of their juvenile offspring; its preservation would greatly benefit from the correct and coordinated management of the residual genetic variability available in the different captive stocks. In this sense, the setup of an efficient parental allocation procedure would allow identifying familiar groups and establishing informed breeding plans, effectively preserving genetic variation. However, being the species tetraploid, the analyses often deal with complex genome architecture and a preliminary evaluation of allele segregation patterns at different chromosomes is necessary to assess whether the species can be considered a pure tetraploid, as previously observed at some loci, or if a more complex situation is present. Here we study the segregation at 14 microsatellites loci in 12 familiar groups. Results support in different families the tetrasomic segregation pattern at 11 markers and the disomic segregation at three markers. The Adriatic sturgeon thus shows a mixed inheritance modality. In this species, and likely in other sturgeons, accurate knowledge of the loci used for paternity analysis is therefore required