Catarina Ginja

Portugal and Spain, with six and 22 officially recognized caprine breeds, encompass 25 % of the European Union goat census. Many of these populations have suffered strong demographic declines because of competition with exotic breeds and the phasing-out of low income rural activities. In this study, we have investigated the consequences of these and other demographic processes on the genetic diversity, population structure and inbreeding levels of Iberian and Atlantic goats. A sample of 975 individuals representing 25 officially recognized breeds from Portugal and Spain, two small populations not officially recognized (Formentera and Ajuí goats) and two ecotypes of the Tinerfeña and Blanca Celtibérica breeds were genotyped with a panel of 20 microsatellite markers. A wide array of population genetics methods was applied to make inferences about the genetic relationships and demography of these caprine populations. Genetic differentiation among Portuguese and Spanish breeds was weak but significant (FST = 0.07; P < 0.001), which is probably the consequence of their short splitting times and extensive gene flow due to transhumance. In contrast, Canarian goats were strongly differentiated because of prolonged geographic isolation. Most populations displayed considerable levels of diversity (mean He = 0.65). High diversity levels and weak population structures are distinctive features of Portuguese and Spanish breeds. In general, these local breeds have a reduced census, but are still important reservoirs of genetic diversity. These findings reinforce the need for the implementation of management and breeding programs based on genetic data in order to minimize inbreeding, maintain overall genetic and allelic diversities and breed identities, while at the same time taking into account the within-breed genetic structure.

ABSTRACT Several studies have investigated the differentiation of mitochondrial DNA in Eurasian, African and American cattle as well as archaeological bovine material. A global survey of these studies shows that haplogroup distributions are more stable in time than in space. All major migrations of cattle have shifted the haplogroup distributions considerably with a reduction of the number of haplogroups and/or an expansion of haplotypes that are rare or absent in the ancestral populations. The most extreme case is the almost exclusive colonization of Africa by the T1 haplogroup, which is rare in Southwest Asian cattle. In contrast, ancient samples invariably show continuity with present-day cattle from the same location. These findings indicate strong maternal founder effects followed by limited maternal gene flow when new territories are colonized. However, effects of adaptation to new environments may also play a role.

Population structure and genetic diversity in the Portuguese native breeds of sheep Algarvia (AL), Badana (BA), Galega Bragançana (GB), Galega Mirandesa (GM), Mondegueira (MO) and Churra da Terra Quente (TQ), as well as the exotic Assaf (AS), were analyzed by typing 25 microsatellite markers in 210 individuals. The markers used exhibited high levels of polymorphism, with means for total and

Population structure and genetic diversity in the Portuguese native breeds of pigs Alentejano (AL), Bísaro (BI), and Malhado de Alcobaça (MA) and the exotic breeds Duroc (DU), Landrace (LR), Large White (LW), and Pietrain were analyzed by typing 22 microsatellite markers in 249 individuals. In general, the markers used were greatly polymorphic, with mean total and effective number of alleles per locus of 10.68 and 4.33, respectively, and an expected heterozygosity of 0.667 across loci. The effective number of alleles per locus and expected heterozygosity were greatest in BI, LR, and AL, and least in DU. Private alleles were found in 9 of the 22 markers analyzed, mostly in AL, but also in the other breeds, with the exception of LW. The proportion of loci not in Hardy-Weinberg equilibrium in each breed analyzed ranged between 0.23 (AL) and 0.41 (BI, LW, and Pietrain), mostly because of a less than expected number of heterozygotes in those loci. With the exception of MA, all breeds showed a significant deficit in heterozygosity (F(IS); P < 0.05), which was more pronounced in BI (F(IS) = 0.175) and AL (F(IS) = 0.139), suggesting that inbreeding is a major concern, especially in these breeds that have gone through a genetic bottleneck in the recent past. The analysis of relationships among breeds, assessed by different methods, indicates that DU and AL are the more distanced breeds relative to the others, with the closest relationship being observed between LR and MA. The degree of differentiation between subpopulations (F(ST)) indicates that 0.184 of the total genetic variability can be attributed to differences among breeds. The analysis of individual distances based on allele sharing indicates that animals of the same breed generally cluster together, but subdivision is observed in the BI and LR breeds. Furthermore, the analysis of population structure indicates there is very little admixture among breeds, with each one being identified with a single ancestral population. The results of this study confirm that native breeds of pigs represent a very interesting reservoir of allelic diversity, even though the current levels of inbreeding raise concerns. Therefore, appropriate conservation efforts should be undertaken, such as adopting strategies aimed at minimizing inbreeding, to avoid further losses of genetic diversity.

Brazilian goat breeds are believed to derive mainly from animals brought by Portuguese settlers since the 16th century. We used microsatellite markers in a sample of 436 animals to study genetic variability and differentiation of the six Portuguese (PT) and six Brazilian (BR) goat breeds currently recognized in the two countries. These breeds were also compared with an outgroup represented by a sample of Alpine (ALP) goats. The effective number of alleles and allelic richness were slightly higher in PT than in BR breeds. The global F ST was nearly 0.11 when PT and BR breeds were considered, with a mean pairwise F ST of about 0.03
among PT breeds, 0.07 among BR breeds and 0.15 between PT and BR breeds. The dendrogram illustrating relationships between populations and the correspondence analysis indicate the existence of two very distinct clusters, corresponding to the countries of origin of the breeds
studied, which are nearly equidistant from the Alpine outgroup. The analysis with structure confirmed the separation between PT and BR breeds but suggests that some BR breeds, especially Grau ́na and Caninde ́, may share a common ancestry with PT breeds. The divergence observed between PT and BR breeds may result from founder effects and genetic drift but could also reflect the introduction in Brazil of goats originating from other regions, e.g., West Africa.

Genetic diversity in and relationships among 26 Creole cattle breeds from 10 American countries were assessed using 19 microsatellites. Heterozygosities, F-statistics estimates, genetic distances, multivariate analyses and assignment tests were performed. The levels of within-breed diversity detected in Creole cattle were considerable and higher than those previously reported for European breeds, but similar to those found in other Latin American breeds. Differences among breeds accounted for 8.4% of the total genetic variability. Most breeds clustered separately when the number of pre-defined populations was 21 (the most probable K value), with the exception of some closely related breeds that shared the same cluster and others that were admixed. Despite the high genetic diversity detected, significant inbreeding was also observed within some breeds, and heterozygote excess was detected in others. These results indicate that Creoles represent important reservoirs of cattle genetic diversity and that appropriate conservation measures should be implemented for these native breeds in order to minimize inbreeding and uncontrolled crossbreeding.

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.

The ancestry of New World cattle was investigated through the analysis of mitochondrial and Y chromosome variation in Creoles from Argentina, Brazil, Mexico, Paraguay and the United States of America. Breeds that influenced the Creoles, such as Iberian native, British and Zebu, were also studied. Creoles showed high mtDNA diversity (H = 0.984 +/- 0.003) with a total of 78 haplotypes, and the European T3 matriline was the most common (72.1%). The African T1a haplogroup was detected (14.6%), as well as the ancestral African-derived AA matriline (11.9%), which was absent in the Iberian breeds. Genetic proximity among Creoles, Iberian and Atlantic Islands breeds was inferred through their sharing of mtDNA haplotypes. Y-haplotype diversity in Creoles was high (H = 0.779 +/- 0.019), with several Y1, Y2 and Y3 haplotypes represented. Iberian patrilines in Creoles were more difficult to infer and were reflected by the presence of H3Y1 and H6Y2. Y-haplotypes confirmed crossbreeding with British cattle, mainly of Hereford with Pampa Chaqueño and Texas Longhorn. Male-mediated Bos indicus introgression into Creoles was found in all populations, except Argentino1 (herd book registered) and Pampa Chaqueño. The detection of the distinct H22Y3 patriline with the INRA189-90 allele in Caracú suggests introduction of bulls directly from West Africa. Further studies of Spanish and African breeds are necessary to elucidate the origins of Creole cattle, and determine the exact source of their African lineages.

Measurements taken on 47 complete and 44 distal fragments of cattle metacarpals from 17 th century AD Carnide, Lisbon, separate into two groups. Comparison with 21 ancient DNA sexed specimens and modern specimens of known sex (seven Barrosã cows and a Barrosã bull), indicates that the Carnide metacarpals probably belonged to both cows and bulls/oxen. We use the 47 complete metacarpals as a " sexed reference sample " in order to find which measurements generally taken by zooarchaeologists on the distal metacarpal help separate males from females. Widths appear to be most useful. The modern Barrosã cattle in our collection, selected for their meat, have wider metacarpals than the ones from Carnide; the latter were perhaps more generalist animals.

In this study, we integrate osteometric and palaeogenetic data to investigate dog variability in the Roman Empire in Iberia and North Africa. Osteometry was used to distinguish the statusddomestic or wild, of approximately 2000 years old Canis remains and to understand to what extent teeth and long bones varied in dogs in the Roman provinces of Mauretania Tingitana, Lusitania and Tarraconensis. High-throughput 454-DNA sequencing technology was used to obtain mitochondrial DNA (mtDNA) sequences from 15 bone and teeth samples. We identified five dog haplotypes from partial sequences of the hypervariable D-loop region. MtDNA haplotypes were grouped into two of the four major clades found in present-day dogs. We detected three clade A haplotypes in 12 samples from Portugal, Spain and Morocco, and a single clade D haplotype in 3 samples from Spain. So far, this is the oldest evidence for the presence of dog clade D in Iberia. It is dated to the late Roman occupation in the 4th-5th cent. AD (ca. 1,600 years ago). Our results confirm the existence of distinct dog morphotypes in Roman times that also harboured distinct genetic lineages. According to our data, dogs from distinct mtDNA lineages (clades A and D) have been continuously bred in the Iberian Peninsula since at least 1600 years ago. Moreover, the sharing of matrilines between dogs from Spain and North Africa may indicate gene flow. Dogs could have been easily transported between these regions by humans along maritime and terrestrial trade routes. These results provide new insights into pre-Roman and Roman domestication practices, confirming selection practices were extensively applied to dogs during the first centuries of our era in the Iberian Peninsula. We show that the greater size variability of teeth length (and consequently cranium) and long bone breadths (and consequently phenotype) of Roman dogs in the Iberian Peninsula, is concomitant with the detection of diverse and rare maternal lineages. This would reflect an intensification of dog breeding and the use of non-local dogs for breeding.

For Iberian ancient dogs, this is the first attempt to successfully apply NGS methods.
Ancient DNA analyses and bioinformatic tools are very powerful to unravel genetic variants from past populations.
Some optimization of the analyses is still necessary to guarantee that the generated consensus sequences are reliable.
Regarding nuclear genes, this is an ongoing study but the preliminary results for the Chalcolithic dog samples show that DNA is poorly preserved for some genes of interest related to coat color, coat texture and body size.
In this way, these preliminary results have shown that NGS Illumina applied on ancient DNA has potential to provide data to further investigations in domestication and evolutionary trajectories of dogs.