Investigating the role of genetic variation in vgll3 and six6 in the domestication of gilthead seabream (Sparus aurata Linnaeus) and European seabass (Dicentrarchus labrax Linnaeus).

Gene function conservation is crucial in molecular ecology, especially for key traits like growth and maturation in teleost fish. The vgll3 and six6 genes are known to influence age-at-maturity in Atlantic salmon, but their impact on other fish species is poorly understood. Here, we investigated the association of vgll3 and six6 in the domestication of gilthead seabream and European seabass, both undergoing selective breeding for growth-related traits in the Mediterranean. We analysed two different sets of samples using two different genotyping approaches. The first dataset comprised farmed and wild populations from Greece, genotyped for SNPs within the two genes ('gene-level genotyping'). The second dataset examined 300-600 k SNPs located in the chromosomes of the two genes, derived from a meta-analysis of a Pool-Seq experiment involving farmed and wild populations distributed widely across the Mediterranean ('chromosome-level genotyping'). The gene-level analysis revealed a statistically significant allele frequency differences between farmed and wild populations on both genes in each species. This finding was partially supported by the chromosome-level analysis, identifying highly differentiated regions may be involved in the domestication process at varying distances from the candidate genes. Noteworthy genomic features were found, such as a CpG island in gilthead seabream and novel candidate genes in European seabass, warranting further investigation. These findings support a putative role of vgll3 and six6 in the maturation and growth of gilthead seabream and European seabass, emphasizing the need for further research on their conserved function.

Genetic Identification and Traceability of Insect Meals.

Insects have been proposed as a rich alternative source of protein for the partial or total replacement of fishmeal in aquaculture. For maximum safety and effectiveness of insect meals, control of the quality composition of these products is considered mandatory. The aim of this study was the genetic analysis of the composition of commercially available insect meals at the species level. Commercially available Hermetia illucens, Tenebrio molitor and Musca domestica individuals, as well as nine insect meals produced from these species, were analyzed. The genetic identification of insects at the species level was based on a COI fragment, and analysis of the insect meals' composition was performed with the processes of cloning and colony PCR. Genetic analysis indicated that the commercially available larvae morphologically identified as Musca domestica belonged to the species Muscina stabulans. In the commercially available insect meals, no other animal species was identified beyond the expected one. However, in the insect meal produced for research purposes, fungal growth was detected. The used methodology, herein, allows for the qualitative genetic identification of insect meals and could be included in the methods of traceability of products containing insects and other animal species.

Prevalence of SOD1 allele associated with degenerative myelopathy in canine population in Greece.

Canine degenerative myelopathy (CDM) is a late-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (c.118G > A). The purpose of this study was to determine the genotype and allele frequencies of this mutation in 108 dogs, mainly in Belgian Malinois and German Shepherd dogs with (CDM-affected group) and without CDM clinical symptoms (control group) in Greece. Genotyping of the c.118G > A mutation was possible by Sanger sequencing and PCR-RFLP. The observed genotype frequencies for the control group were 89.4% for the homozygous (G/G), 9.6% for the heterozygous (A/G), and 0.96% for the homozygous mutant (A/A) allele. The mutant allele was not common in the Belgian Malinois dogs (allele frequency = 0.029), but quite common in the German Shepherd dogs (allele frequency = 0.138). In the CDM affected group, all 4 dogs were homozygous for the mutant allele. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium. A strong but not statistically significant association between the mutant allele and CDM was observed. A previously identified deletion upstream of the mutation of interest was found at a high frequency (0.361) in the population.

DNA barcoding identification of Greek freshwater fishes.

Biodiversity is a key factor for the functioning and efficiency of an ecosystem. Greece, though covering a relatively small surface area, hosts a great deal of species diversity. This is especially true for freshwater fishes. In recent years, the traditional methods of species identification have been supplemented by the use of molecular markers. The present study therefore aims to extensively produce DNA barcodes for Greek freshwater fish species and investigate thoroughly if the presently accepted species classification is in agreement with molecular data. A 624-bases long fragment of the COI gene was sequenced, from 406 freshwater fish specimens belonging to 24 genera and originating from 18 lake and river sites. These sequences were used along with 596 sequences from the same genera, recovered from BOLD, for the construction of phylogenetic trees and the estimation of genetic distances between individuals. In total, 1002 sequences belonging to 72 species were analyzed. The method was found to be effective for 55 of 72 studied species. 17 closely related species with low interspecific genetic distances were observed, for which further study is proposed. It should also be noted that, in four cases, cryptic diversity was observed, where groups originally identified as one species exhibited genetic distance great enough to be separated into discrete species. Region specific haplotypes were also detected within populations of 14 species, giving the possibility to identify even the geographic origin of a species. Our findings are discussed in the light of the rich history of the Balkan peninsula and provide a significant steppingstone for the further study of Greek and European freshwater fish biodiversity.

Multisource noninvasive genetics of brown bears (Ursus arctos) in Greece reveals a highly structured population and a new matrilineal contact zone in southern Europe.

In human-dominated landscapes, connectivity is crucial for maintaining demographically stable mammalian populations. Here, we provide a comprehensive noninvasive genetic study for the brown bear population in the Hellenic Peninsula. We analyze its population structuring and connectivity, estimate its population size throughout its distribution, and describe its phylogeography in detail for the first time. Our results, based on 150 multilocus genotypes and on 244-bp sequences of the mtDNA control region, show the population is comprised by three highly differentiated genetic clusters, consistent with geographical populations of Pindos, Peristeri, and Rhodope. By detecting two male bears with Rhodopean ancestry in the western demes, we provide strong evidence for the ongoing genetic connectivity of the geographically fragmented eastern and western distributions, which suggests connectivity of the larger East Balkan and Pindos-Dinara populations. Total effective population size (N e) was estimated to be 199 individuals, and total combined population size (N C) was 499, with each cluster showing a relatively high level of genetic variability, suggesting that migration has been sufficient to counteract genetic erosion. The mtNDA results were congruent with the microsatellite data, and the three genetic clusters were matched predominantly with an equal number of mtDNA haplotypes that belong to the brown bear Western mitochondrial lineage (Clade 1), with two haplotypes being globally new and endemic. The detection of a fourth haplotype that belongs to the Eastern lineage (Clade 3a1) in three bears from the western distribution places the southernmost secondary contact zone between the Eastern and Western lineages in Greece and generates new hypotheses about postglacial maxima migration routes. This work indicates that the genetic composition and diversity of Europe's low-latitude fringe population are the outcome of ancient and historical events and highlight its importance for the connectivity and long-term persistence of the species in the Balkans.

Genome-wide analysis clarifies the population genetic structure of wild gilthead sea bream (Sparus aurata).

Gilthead sea bream is an important target for both recreational and commercial fishing in Europe, where it is also one of the most important cultured fish. Its distribution ranges from the Mediterranean to the African and European coasts of the North-East Atlantic. Until now, the population genetic structure of this species in the wild has largely been studied using microsatellite DNA markers, with minimal genetic differentiation being detected. In this geographically widespread study, 958 wild gilthead sea bream from 23 locations within the Mediterranean Sea and Atlantic Ocean were genotyped at 1159 genome-wide SNP markers by RAD sequencing. Outlier analyses identified 18 loci potentially under selection. Neutral marker analyses identified weak subdivision into three genetic clusters: Atlantic, West, and East Mediterranean. The latter group could be further subdivided into an Ionian/Adriatic and an Aegean group using the outlier markers alone. Seascape analysis suggested that this differentiation was mainly due to difference in salinity, this being also supported by preliminary genomic functional analysis. These results are of fundamental importance for the development of proper management of this species in the wild and are a first step toward the study of the potential genetic impact of the sea bream aquaculture industry.

Genetic structure and divergence of tench Tinca tinca European populations.

The tench Tinca tinca is a freshwater species with human-mediated translocations, aquaculture interest and limited information on its genetic structure. mtDNA sequencing analysis of control region and two genes in 50 individuals from five European populations identified two phylogroups, with greater variability than that reported until now, and a hybridization zone in the Danube River region. Restriction analyses of additional samples reveal the complicated genetic structure characteristics of tench's wild and translocated populations, supporting future breeding practices.

The genetic population structure and temporal genetic stability of gilthead sea bream Sparus aurata populations in the Aegean and Ionian Seas, using microsatellite DNA markers.

We examined 662 gilthead sea bream Sparus aurata from wild samples of the species in the Aegean and Ionian Seas, using 20 EST-linked microsatellite markers, in three multiplex panels, as well as seven anonymous loci. Most of the markers were revealed to be highly polymorphic. We found low genetic differentiation between the sampling stations/areas with total FST 0.002 (P < 0.05). Based on comparison of five temporal samples, our results indicate genetic data consistency over time for all tested samples, pointing to stable populations, despite reported repeated escape events. Our results confirm the genetic population structure previously observed in these specific areas, using by far more markers than in previous studies in both coding and non-coding DNA loci. The limited genetic structure and the temporal genetic stability indicate neither major genetic differentiation of local populations by geographic isolation nor influence from anthropogenic factors. These results provide a baseline for future reference in any management programme of both wild and farmed population of S. aurata as well as of other aquaculture species with a potential introgression among farmed and wild populations.

Genetic analyses of brown hare (Lepus europaeus) support limited migration and translocation of Greek populations.

Numerous studies have shown that the phylogeography of many species, including European brown hare, has been affected by the climatic oscillations of the Pleistocene. During this period the Balkans acted as a major refugium offering habitable conditions for many species. However, few studies have focused on the specific role of the Greek peninsula in the phylogeographic history of species in this southernmost margin of Balkans. We, therefore analyzed a 528 bp fragment of the D-loop region of mitochondrial DNA in 154 wild brown hare individuals from unsampled areas from both mainland and island Greece and compared it to 310 available brown hare sequences (including 110 Greek samples). Newly identified haplotypes show characteristic distribution in specific Greek areas reinforcing the theory that Greece can be considered as a subrefuge within Balkans for a number of species, with several "refugia within refugia" spots, holding significant genetic diversity. No haplotypes from wild Greek individuals clustered with the Central and Western Europe group revealing a minimal contribution of this area to the colonization of central Europe. One hundred and ten reared brown hares were also analyzed to elucidate the impact of introductions on local populations. Most of these samples presented close genetic affinity with haplotypes from Central and Western Europe indicating that farms in Greece use breeders from those areas. Therefore, despite human translocation of individuals, the genetic structure of brown hare has mostly been influenced by paleoclimatic conditions and minimally by human actions.

Greece: a Balkan subrefuge for a remnant red deer (cervus elaphus) population.

A number of phylogeographic studies have revealed the existence of multiple ice age refugia within the Balkan Peninsula, marking it as a biodiversity hotspot. Greece has been reported to harbor genetically differentiated lineages from the rest of Balkans for a number of mammal species. We therefore searched for distinct red deer lineages in Greece, by analyzing 78 samples originating from its last population in Parnitha Mountain (Central Greece). Additionally, we tested the impact of human-induced practices on this population. The presence of 2 discrete mtDNA lineages was inferred: 1) an abundant one not previously sampled in the Balkans and 2) a more restricted one shared with other Balkan populations, possibly the result of successful translocations of Eastern European individuals. Microsatellite-based analyses of 14 loci strongly support the existence of 2 subpopulations with relative frequencies similar to mitochondrial analyses. This study stresses the biogeographic importance of Central Greece as a separate Last Glacial Maximum period refugium within the Balkans. It also delineates the possible effects that recent translocations of red deer populations had on the genetic structuring within Parnitha. We suggest that the Greek red deer population of Parnitha is genetically distinct, and restocking programs should take this genetic evidence into consideration.